CN108014101B - Application of the erucic acid in the drug of preparation prevention or treatment Flu-A - Google Patents
Application of the erucic acid in the drug of preparation prevention or treatment Flu-A Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
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- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
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Abstract
The present invention provides new application of the erucic acid in terms of preparation is for preventing or treating Flu-A drug.The present invention also provides a kind of for preventing or treating the drug of Flu-A, and the active constituent of the drug includes erucic acid.The inflammatory reaction that present inventor has found that erucic acid replicates influenza A virus and mediates has significant inhibitory activity.It is confirmed using liquid-phase chip technology, erucic acid can obviously inhibit the unconventionality expression of the H1N1 virus infection A549 cellular inflammation factor and in dose-dependence；It is confirmed using Western blot, erucic acid can inhibit with the activation of host inflammation associated signal paths and in dose-dependence；It is confirmed using Annexin V-FITC/PI double labelling flow cytometry, the Apoptosis of Host Cells that erucic acid can obviously inhibit influenza H1N1 to induce is damaged and is in dose-dependence.It is confirmed by zoopery, lung inflammation and the apoptosis damage that erucic acid can obviously inhibit Influenza virus H1N1 to induce effectively extend the life span of mouse.
Description
Technical field
The invention belongs to field of medicaments, it is related to a kind of application of natural products in terms of preparing Tamiflu, especially relates to
And erucic acid is in preparation prevention or the new application for the treatment of Flu-A drug.
Background technique
Influenza (abbreviation influenza) is the acute respiratory disease as caused by influenza virus, is had highly infectious.
Global annual flu episode rate is about 5%-10% in adult, is about 20%-30% in children, in people at highest risk such as baby children
It is easily caused in youngster, the elderly or chronic in hospital and dead.The annual influenza in the whole world cause about 3,000,000 to 5,000,000 it is tight
Weight disease and about 250,000 to 500,000 death, seriously threaten the life and health of the mankind, and cause huge economic losses.The stream of people is susceptible
Poison is divided into first, second, the third three types, and wherein Flu-A threatens the mankind maximum, and H1N1 is most important seasonal Flu-A disease
Poison.The most effectual way of prevention and control influenza spread is vaccine inoculation, but since the antigenic variation ability of influenza virus is strong,
Only for popular influenza subtype strain, the novel influenza generated for antigenic drift and variation infects not for the production of vaccine
Generate the protective effect of body.Therefore, influenza vaccines flu-prevention has certain hysteresis quality, and protective rate is also limited.It uses at present
In the chemicals for the treatment of of influenza, there are two main classes: adamantane amine (amantadine and Rimantadine) and neuraminidase inhibit
Agent (Oseltamivir and zanamivir) need to usually be administered in disease early stage (after symptom appearance in 48 hours), and due to acting on target
Point is single to have there is drug resistance, significantly impacts curative effect.Therefore, clinical to need to research and develop new Tamiflu.
During influenza infection carries out self-replacation using host, it is anti-that host starts inherent immunity reaction generation
Virokine (such as interferon) and inflammatory reaction are to inhibit the virus invaded.The inflammatory reaction of appropriateness is conducive to the clear of influenza virus
It removes.But excessive inflammatory response can cause " inflammatory factor storm ", lead to the serious tissue damage of body, this is that influenza leads to death
One of the main reason for.Therefore, to inhibit influenza virus duplication and excessive inflammatory response to have become and grind as the medicament research and development of target
Study carefully hot spot.Also modulate host inherent immunity reacts relevant signal path and inflammatory factor while such drug is directly antiviral
Secretion, therefore it is significant in efficacy and be not easy drug resistance.
Erucic acid (Erucic acid) is widespread in nature, and is refining to obtain mustard with distillation by the seed of leaf mustard is fermented
Seed oil and erucic acid, then obtained through separating-purifying.Or using rape oil or oil foot as raw material, with traditional method (saponification acidolysis；Acidification
Hydrolysis or pressurized hydrolysis) rape oil fatty acid is made in hydrolysis, then isolates erucic acid from rape oil fatty acid.To fatty acid mixed using cold
Freeze, squeeze, after initial gross separation, then purer erucic acid can be obtained using rectification under vacuum.But about erucic acid in suppressing virus replication and
Effect in terms of the inflammatory reaction for inhibiting influenza A virus to mediate did not appeared in the newspapers.
Summary of the invention
The purpose of the present invention is to provide new application of the erucic acid in terms of preparation is for preventing or treating Flu-A drug.
For this purpose, the application the present invention provides erucic acid in the drug of preparation prevention or treatment Flu-A.
The erucic acid has the following structure formula:
The medicine of inflammatory reaction the present invention also provides erucic acid in preparation for inhibiting influenza A virus to replicate and its mediate
Application in object.
The present invention also provides a kind of for preventing or treating the drug of Flu-A, and the active constituent of the drug includes mustard
Acid, erucic acid can be used as sole active agent, can also combine with the substance of other medicinal licenses.The drug can further comprise pharmacy
Upper acceptable carrier or auxiliary material, can be prepared into pharmaceutically acceptable dosage form, dosage form is, for example, tablet, ebonite as needed
The various peroral dosage forms such as capsule, soft capsule, granule, dripping pill.Required pharmaceutically acceptable carrier or auxiliary material is specific such as pharmacy
Upper common diluent and absorbent, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urine
Element, calcium carbonate, white bole, microcrystalline cellulose, alumina silicate etc.；Pharmaceutically common wetting agent and adhesive such as water, glycerol, gather
Ethylene glycol, ethyl alcohol, propyl alcohol, starch slurry, dextrin, syrup, honey, glucose solution, mucialga of arabic gummy, gelatine size, carboxymethyl are fine
Tie up plain sodium, lac, methylcellulose, potassium phosphate, polyvinylpyrrolidone etc.；Pharmaceutically common disintegrating agent, such as dry shallow lake
Powder, alginate, agar powder, laminaran, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, polyoxyethylensorbitan fatty acid
Ester, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc.；Disintegration inhibitor, for example, sucrose, glyceryl tristearate,
Cocoa butter, hydrogenated oil and fat etc.；Lubricant, for example, it is talcum powder, silica, cornstarch, stearate, boric acid, atoleine, poly-
Ethylene glycol etc..It is no longer enumerated about pharmaceutically acceptable carrier or auxiliary material, those of ordinary skill in the art can be according to institute
The common knowledge of grasp is specifically chosen.
The drug for the inflammatory reaction that the present invention also provides a kind of for inhibiting influenza A virus and its mediation, the drug
Active constituent include erucic acid, erucic acid can be used as sole active agent, can also combine with the substance of other medicinal licenses.The drug
It can further comprise pharmaceutically acceptable carrier or auxiliary material, can be prepared into pharmaceutically acceptable dosage form, dosage form as needed
The for example, various peroral dosage forms such as tablet, hard capsule, soft capsule, granule, dripping pill.Required pharmaceutically acceptable carrier or
Auxiliary material, it is specific such as pharmaceutically common diluent and absorbent, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose,
Sodium chloride, glucose, urea, calcium carbonate, white bole, microcrystalline cellulose, alumina silicate etc.；Pharmaceutically common wetting agent and bonding
Agent, as water, glycerol, polyethylene glycol, ethyl alcohol, propyl alcohol, starch slurry, dextrin, syrup, honey, glucose solution, mucialga of arabic gummy,
Gelatine size, sodium carboxymethylcellulose, lac, methylcellulose, potassium phosphate, polyvinylpyrrolidone etc.；Pharmaceutically commonly collapse
Solve agent, for example, dry starch, alginate, agar powder, laminaran, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene,
Sorbitan fatty acid ester, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc.；Disintegration inhibitor, such as sucrose,
Glyceryl tristearate, cocoa butter, hydrogenated oil and fat etc.；Lubricant, for example, talcum powder, silica, cornstarch, stearate,
Boric acid, atoleine, polyethylene glycol etc..It is no longer enumerated about pharmaceutically acceptable carrier or auxiliary material, this field is common
Technical staff can be specifically chosen according to the common knowledge grasped.Present inventor has found erucic acid to influenza A virus
Duplication and its inflammatory reaction mediated have significant inhibitory activity.Present inventor uses mdck cell lesion method, supernatant
Progeny virus Inhibition test confirms that erucic acid can obviously inhibit the self-replication of H1N1 virus；It is confirmed using Suspension array technique,
Inflammatory factor IL-1beta, IL-6, IL-21, IP-10, IL-17A, IL- that erucic acid can obviously inhibit influenza virus to induce
17F, IFN-γ, TNF-α unconventionality expression and be in dose-dependence；It is confirmed using Western blot, erucic acid can inhibit and place
The activation of main inflammation associated signal paths NF- κ B, P38MAPK, ERK1/2MAPK, P-JNK, P-AKT are in dose-dependence.
Confirm that erucic acid obviously can inhibit Influenza virus H1N1 to lure by zoopery (infection H1N1 virus mouse experiment in vivo)
The injury of lungs and inflammation led extend the mouse survival time.Erucic acid is expected to novel anti-as a kind of safely and effectively mechanism of action
Influenza newtype drug.
Compared with existing drug, has outstanding advantage: 1, erucic acid action target using erucic acid preparation anti influenza newtype drug
Both for virus, also for host cell, therefore it is not likely to produce drug resistance；2, erucic acid is without obvious toxic-side effects；3, erucic acid is plant list
Body ingredient, structure is clear, and chemical property is stablized, and is easy to quality control；4, erucic acid abundance, it is cheap and easy to get, big life can be met
Production demand.
Detailed description of the invention
Fig. 1 shows erucic acid to the inhibiting effect of influenza A/PR8/3/4 (H1N1) progeny virus.
Fig. 2 shows erucic acid to influenza A/PR8/3/4 (H1N1) (MOI=0.1) induction people A549 cell-signaling pathways activation
Effect.
Fig. 3 shows to detect the infection induced place of erucic acid infected by influenza in protein level using Bio-plex Suspension array technique
The effect of chief cell inflammatory response.
Fig. 4 shows that Flow cytometry erucic acid damages the Apoptosis of Host Cells that influenza A/PR8/3/4 (H1N1) is induced
Inhibiting effect.
Fig. 5 shows in virus infection A549 cell, detects erucic acid Apoptosis using western blot technology
The effect of molecule pro-caspase3 and substrate PARP.
Fig. 6 shows the protective effect of interior evaluating erucic acid influenza virus infected；Wherein A are as follows: erucic acid is to infection disease
The Lung Exponent of malicious mouse influences；B are as follows: adjustment effect of the erucic acid to protein concentration in virus infection mouse bronchoalveolar lavage fluid；C are as follows:
Adjustment effect of the erucic acid to IL-6 in the homogenate of virus infection mouse lung tissue；D are as follows: erucic acid is homogenized virus infection mouse lung tissue
The adjustment effect of middle TNF-α；E are as follows: adjustment effect of the erucic acid to MCP-1；F are as follows: adjustment effect of the erucic acid to MIP-1；G are as follows: mustard
Adjustment effect of the acid to GM-CSF；H are as follows: effect of the erucic acid to virus infection mouse.
Specific embodiment
Technical scheme is described further with reference to the accompanying drawing.If instrument or reagent are not in embodiment
It illustrates, is that the conventional products obtained can be bought by market using this field conventional reagent or instrument.
One erucic acid of embodiment causes the inhibiting effect of cytopathy to common influenza virus
1, material: cell and virus
The mdck cell (less than 25 generations) of low algebra is purchased from ATCC, is stored in this laboratory；A/PR/8/34(H1N1),A/
GZ/GIRD07/09 (H1N1), A/HK/8/68 (H3N2), A/HK/Y280/97 (H9N2), B/Lee/1940 (FluB) are stored in
This laboratory.
2, reagent:
DMEM/DF12 (1:1) culture medium is purchased from Gibco company；MTT is purchased from sigma company, and 5mg/mL MTT solution=
MTT+100mL DMEM/DF12 (1:1) culture medium of 50mg；Virus-culturing fluid (the TPCK pancreatin of the 1.5ug/mL containing final concentration):
The 1mg/mLTPCK pancreatin of 100mL DMEM/DF12 (1:1) culture solution+150uL, i.e., with i.e. use；Erucic acid is from Radix Isatidis
In it is isolated, be white powder, preparation method reference literature [modern medicines and clinical, 2011,26 (5): 381-383] carries out,
It can be bought by market or be obtained using existing other methods.
3, inhibiting effect of the erucic acid to common influenza virus
1) 96 orifice plate MDCK cell monolayers are washed 1 time with PBS；
2) 100 TCID50 virus liquids are added, 100 holes μ L/, 37 DEG C are incubated for 2 hours；
3) virus incubation liquid is discarded, the Virus culture of the TPCK pancreatin with the 1.5ug/mL containing final concentration is added in pharmaceutical intervention group
Erucic acid (1000ug/ml, 500ug/ml, 250ug/ml, 125ug/ml, 62.5ug/ml, 31.25ug/ of liquid multiple proportions gradient dilution
ml,15.625ug/ml).37 DEG C culture 48 hours after observe result；
4) observation caliber such as following table
6 grade standard of cytopathy (CPE) caused by 1 virus of table
5) IC of CPE method50Formula
Drug effect inhibiting rate=1- of CPE is averaged every hole extent of disease (%)
CPE distance is than=(>50% inhibiting rate -50%)/(>50% inhibiting rate -<50% inhibiting rate)
The IC of CPE method50=less than the drug concentration X2 of 50% inhibiting rateDistance than
As shown in the following Table 2, as a result illustrate that erucic acid can effectively inhibit influenza virus A/PR/8/34 (H1N1), A/GZ/
Cytopathy caused by GIRD07/09 (H1N1), A/HK/8/68 (H3N2).
Table 2
Inhibiting effect of two erucic acid of embodiment to influenza A/PR8/3/4 (H1N1) cells and supernatant generation of neutrons virus
1, material: cell and virus
A549 cell is purchased from ATCC, is stored in this laboratory
2, inhibiting effect of the erucic acid to influenza A/PR8/3/4 (H1N1) progeny virus
1) 6 orifice plates single layer A549 cell discards former culture medium, and PBS is added and washes twice；
2) serum-free DMEM/DF12 (1:1) culture medium for containing influenza virus A/PR8/3/4 (H1N1), 37 DEG C of absorption are added
Cell 2h；
3) cell is washed with PBS to remove unadsorbed virus；
4) mode of drug is used, erucic acid (100 μ g/ml, 200 μ g/ml, 300 of various concentration are added in pharmaceutical intervention group
μ g/ml), the erucic acid of 200 μ g/ml is added in drug alone group, and the A549 cell of drug alone group is adsorbed without influenza virus, and 37 DEG C
Culture 24 hours；
5) cells and supernatant is collected；
6) 96 orifice plate MDCK cell monolayers are washed 1 time with PBS；
7) 100 hole μ L/ of cells and supernatant collected above is added, 37 DEG C are incubated for 2 hours；
8) 96 orifice plates sop up supernatant, and 100 hole μ L/ of virus-culturing fluid of the TPCK pancreatin of the 1.5ug/mL containing final concentration is added,
37 DEG C are cultivated 48 hours；
9) observation is as a result, immediately arrive at TCID50。
As a result as shown in Figure 1, erucic acid can effectively inhibit influenza virus A/PR/8/34 (H1N1) cells and supernatant generation of neutrons
The titre of virus.
The effect of three erucic acid infected by influenza A/PR8/3/4 (H1N1) of embodiment induction host signal access
1, chemical reagent and antibody
Sodium chloride (NaCl), potassium chloride (KCl), potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4
12H2O), glycine Glycine, tween Tween-20 are purchased from Guangzhou Chemical Reagent Factory；Tris base is public purchased from Ameresco
Department；Dodecyl sodium sulfate (SDS) is purchased from U.S. sigma company；Ammonium Persulfate 98.5 (APS) is purchased from U.S. Ameresco company；
TEMED (N,N methylene bis acrylamide) is purchased from U.S. Bio-rad company；RIPA lysate is purchased from U.S. Thermo company；
PMSF is purchased from U.S. sigma company；Cocktail (protease inhibitors) is purchased from U.S. sigma company；BCA determination of protein concentration
Kit is purchased from U.S. Thermo company；Easysee Western marker (20-90kDa) is purchased from Chinese T ransgen
Biotech company；Blue plus II protein marker is purchased from Chinese T ransgen Biotech company；Pvdf membrane purchase
From Life science company, the U.S.；Skimmed milk power is purchased from Gibco company；Fetal calf serum is purchased from Gibco company；Rabbit-anti people p-
P65 antibody is purchased from U.S. CST company；Rabbit-anti people's p65 antibody is purchased from U.S. CST company；Rabbit-anti people's p-p38 antibody is purchased from the U.S.
CST company；Rabbit-anti people's p38 antibody is purchased from U.S. CST company；
Rabbit-anti people's p-pERK antibody is purchased from U.S. CST company；Rabbit-anti people's ERK antibody is purchased from U.S. CST company；Rabbit-anti people p-
JNK antibody is purchased from U.S. CST company；Rabbit-anti people's JNK antibody is purchased from U.S. CST company；Rabbit-anti people's p-AKT antibody is purchased from the U.S.
CST company；Rabbit-anti people's AKT antibody is purchased from U.S. CST company；Rabbit-anti people's GAPDH antibody is purchased from U.S. CST company；Goat-anti rabbit two
It is anti-to be purchased from U.S. Earthox company.
2, erucic acid inhibits the activation of influenza inducing host cell signal path
1) cell is laid on 6 orifice plates by the density for adjusting A549 cell, is placed in incubator, after culture 12h cell is adherent
For use；
2) setting of experimental group is carried out, comprising: normal group, influenza virus group, low-dose drugs intervention group (influenza virus+
Erucic acid (100 μ g/ml)), middle dosage pharmaceutical intervention group (influenza virus+erucic acid (200 μ g/ml)), high dose medicament intervention group (stream
Influenza Virus+erucic acid (300 μ g/ml)), drug alone group (erucic acid (300 μ g/ml))；
3) A549 cell is taken out, discards culture medium, PBS is added and washes twice, is then added and contains influenza virus A/PR8/
DMEM/DF12 (1:1) the culture medium 1ml/ hole adherent cell 2h of 3/4 (H1N1) (MOI=0.1) serum-free；
4) unadsorbed extra virus is washed away with PBS, erucic acid (100 μ g/ml, 200 of various concentration are added in pharmaceutical intervention group
μ g/ml, 300 μ g/ml), the erucic acid of 200 μ g/ml is added in drug alone group.37 DEG C are continued culture 24 hours；
5) cell 6 orifice plates are placed on ice, discard culture supernatant, and washed twice with cold PBS；Washing finishes, rapidly
130ul cell pyrolysis liquid RIPA is added (containing protease inhibitors cocktail, with cell pyrolysis liquid RIPA volume ratio (V/
V it is) 1:100 addition and the PMSF of final concentration of 10uM), extracts total protein of cell；
6) 1.5mlEP pipe is marked to each group EP pipe for being placed in and being transferred to cell lysate marked on ice, and
30min is shaken on shaking table, and albumen is allowed sufficiently to crack；
7) centrifuge that cell lysate is moved to 4 DEG C of pre-coolings carries out 13000rpm × 15min centrifugation；
8) centrifugation is finished, and supernatant is carried out packing and is stored in -80 DEG C；
9) protein concentration of extraction is measured, carries out configuration standard product by BCA protein detection kit specification, so
The BCA detection hole working solution 200ul/ (configuration work liquid A:B=1:50) is hole-specifically added afterwards, 37 DEG C of incubation 30min after mixing take out
96 orifice plates are cooled to room temperature, and measure OD value in absorbance 562nm in microplate reader.Standard curve is drawn, obtains protein concentration.
10) preparation of sds page:
According to recipe configuration 10%SDS polyacrylamide gel electrophoresis separation gel: 4ml ddH2O+3.3ml 30%ACr
(acrylamide)+2.5ml 1.5M Tris.HCl (PH=8.8)+0.1ml 10%SDS+0.1ml 10%APS (ammonium persulfate)
Separation gel is quickly perfused in+0.004ml TEMED between separation of glasses plate after mixing well, perfusion, which finishes, is added ddH20 on upper layer
Separation glue liquid surface is sealed, waits the condensation of 30min separation gel intact；It is dense according to recipe configuration 5%SDS polyacrylamide gel electrophoresis
Contracting glue: 3.4ml ddH2O+0.83ml 30%ACr (acrylamide)+0.63ml 1M Tris.HCl (PH=6.8)+0.05ml
10%SDS+0.05ml 10%APS (ammonium persulfate)+0.005ml TEMED is quickly perfused between glass plate dense after mixing well
Contracting glue is perfused and finishes insertion comb, and excludes bubble, waits 30min concentration gelling knot intact.
11) it is loaded:
1:4 ratio is pressed with 5 times of concentration SDS polyacrylate hydrogel sample loading buffers (5Xloading buffer) and sample (20 μ g)
Example mixing, 95 DEG C of denaturation 10min, being immediately passed to cooled on ice 10min prevents protein renaturation.It is centrifuged 3000rpm × 1min, hole-specifically
Sample-adding；
12) electrophoresis:
With Bio-Rad vertical electrophoresis apparatus, 80V constant pressure electrophoresis about (1.5-2) h reaches separation gel bottom to bromophenol blue indicator
Stop electrophoresis when portion；
13) protein transferring film:
Pvdf membrane first impregnates 15min with methanol.Gel is removed after electrophoresis, extra Blank gel is cut off, after trimming
6 Whatman3M filter paper are cut by its size.Neat postposition is stacked by 3 filter paper-gel-filter paper of pvdf membrane -3 sequences
Turn on negative plate in wet, is turned with the bubble between exhaust bubble plate venting gel and pvdf membrane in freezer with the constant current of 380mA
Film 2h.After transferring film, takes out pvdf membrane and be placed in 5% skimmed milk power (5%milk/TBST) and in jog on room temperature shaker
2h；
14) configure 5%BSA/TBST, by purchased from antibody NF-B, P38MAPK of CST company, ERK1/2MAPK, P-JNK,
The corresponding dilution proportion antibody of P-AKT specification, is added to and has closed on the pvdf membrane finished, be placed in Cool Room 4 DEG C and be incubated for
Night；
15) it second day, takes out pvdf membrane and restores to room temperature, remove primary antibody and washed film 3 times, 5min/ times with washing lotion TBST.
The goat-anti rabbit secondary antibody with horseradish peroxidase (HRP) coupling that (V/V) 1:1000 by volume is diluted, room is then added
Temperature is incubated for 1h.It removes secondary antibody and is washed film 3 times, 5min/ times with washing lotion TBST；
16) film is taken out, the luminous color developing agent of ECL immunofluorescence is added in drip-dry moisture on filter paper；
17) tabletting is carried out in darkroom, and in developing liquid developing and the fixed film of fixing solution；
18) interpretation of result and arrangement.
As a result arrange after as shown in Fig. 2, influenza A/PR8/3/4 (H1N1) infection A549 cell for 24 hours after, cell-signaling pathways
NF-B, P38MAPK, ERK1/2MAPK, P-JNK, P-AKT are obviously activated.Erucic acid obviously inhibits influenza A/PR8/3/4 (H1N1) to lure
Lead people A549 cell-signaling pathways NF- κ B, P38MAPK, the activation of signal path.
Example IV liquid-phase chip technology the protein level infection induced host cell of detection erucic acid infected by influenza because
The effect of son secretion
1,6 orifice plates single layer A549 cell discards archaeocyte culture medium, and PBS is added and washes twice, and is then added containing influenza disease
DMEM/DF12 (1:1) culture medium adherent cell of malicious A/PR8/3/4 (H1N1) serum-free.After 2h, cell is washed with PBS and is removed
Go unadsorbed virus.If control group (for normal A549 cell), viral group, virus+pharmaceutical intervention group, drug alone group；Its
In, virus+pharmaceutical intervention group uses mode of drug, and erucic acid (100 μ g/ml, 200 μ of various concentration are added in pharmaceutical intervention group
g/ml,300μg/ml)；Viral group is not added with pharmaceutical intervention.Drug alone group, A549 cell are added 200 without viruses adsorption
The erucic acid of μ g/ml.After continuing culture 24 hours, collects cells and supernatant and managed in 1.5mlEP.Then, be placed in 4 DEG C pre-cooling from
The centrifugation of 13000rpm x 15min is carried out in scheming.Centrifugation is finished, and supernatant is carried out aliquot packing, multigelation is avoided, freezes
In -80 ° of refrigerators, used when to be detected.
2, cytokine content in Bio-plex Suspension array technique detection cell conditioned medium:
1) it takes out sample to be tested slowly to be thawed on ice, takes out Bio-Plex suspension chip inspecting reagent unit (Bio
Rad company), make it restore to room temperature；
2) on setting experiment flat underside standard sample wells, blank well and sample well position, standard sample wells and blank well each 2
Multiple holes；
3) detection buffer assay is added in (Bio-Plex suspension chip inspecting reagent unit) ratio to specifications
Buffer is diluted antibody microballoon, sufficient vortex homogeneous magnetic microballoon, and every hole 50ul microballoon mixed liquor is added in flat underside；
4) 100ul dcq buffer liquid wash buffer is added in every hole, and flat underside is placed on magnetic frame and carries out washing 2
It is secondary；
5) it is loaded: the standard items being serially diluted, ready sample is added separately to standard sample wells, the sample of flat underside
In sample wells, assay buffer is added in blank well, with sealed membrane sealing plate；
6) it is incubated for: being wrapped and be protected from light with aluminium foil, flat underside is incubated at room temperature 30min in 96 plate shaker 300rpm；
7) wash: incubation, which finishes for flat underside to be placed on magnetic frame, discards liquid.100ul wash is added in every hole
Buffer, and flat underside is placed on magnetic frame and discards liquid, it repeats this process and washs 3 times；
8) it plus detects antibody: shaking up detection antibody using preceding sufficient vortex, will test antibody, ratio is added to specifications
To detection antibody diluent in, vortex uniform cell factor IL-1beta, IL-6, IL-21, IP-10, IL-17A, IL-17F,
The detection antibody of IFN-γ, TNF-α is added 25ul in the every hole of flat underside and detects antibody.At room temperature, flat underside is placed in shaking table
300rpm is incubated for 30min；
9) wash: incubation, which finishes for flat underside to be placed on magnetic frame, discards liquid, and 100ul wash is added in every hole
buffer.Flat underside is placed on magnetic frame and discards wash buffer, this process is repeated and washs 3 times；
10) Streptavidin of 50ul fluorescein PE label is added in the every hole of flat underside, room temperature is in 96 plate shakers
300rpm is incubated for 10min；
11) wash: incubation, which finishes for flat underside to be placed on magnetic frame, discards liquid, and 100ul wash is added in every hole
Buffer is placed on magnetic frame and discards wash buffer, washes repeatedly 3 detections；
12) 125ul is added in the every hole of flat underside and detects buffer assay buffer, be protected from light rocker 1100rpm* at room temperature
It 30 seconds, is detected with Bio-plex 200；
As a result inflammatory factor (the IL- for obviously inhibiting influenza virus to induce as shown in figure each in Fig. 3, after erucic acid intervention
1beta, IL-6, IL-21, IP-10, IL-17A, IL-17F, IFN-γ, TNF-α) level.Prompt erucic acid may pass through inhibition
Influenza infection induces the excessive inflammation response of body to play the effect for the treatment of influenza infection.
Embodiment five is using Annexin V-FITC/PI double labelling Flow cytometry erucic acid to influenza A/PR8/3/4
(H1N1) inhibiting effect of the Apoptosis of Host Cells damage of (MOI=0.1) induction
1, A549 cell kind is long to single layer in 6 orifice plates, discards former culture medium, PBS is added and washes twice, then plus containing influenza
Serum-free DMEM/DF12 (1:1) the culture medium adherent cell 2h of viral A/PR8/3/4 (H1N1) (MOI=0.1).It is washed with PBS
Cell removes unadsorbed extra virus.If control group (for normal A549 cell), viral group, pharmaceutical intervention group, drug alone
Group；Wherein, pharmaceutical intervention group uses mode of drug, and erucic acid (100 μ g/ml, 200 μ of various concentration are added in pharmaceutical intervention group
g/ml,300μg/ml)；Viral group is not added with pharmaceutical intervention.Drug alone group, A549 cell are added 200 without viruses adsorption
The erucic acid of μ g/ml.Continue 37 DEG C to cultivate 24 hours.
2, the cell collected in 6 orifice plates carries out streaming Annexin V (being purchased from affymetrix eBioScience company)
1) cell that analysis collects in 6 orifice plates in each experimental group supernatant is managed in 1.5mlEP mark, room temperature 1000rpm 3min from
The heart, PBS are washed 1 time, and 1X Annexin V Binding Buffer is then added and washs 1 time, room temperature 1000rpm 3min centrifugation,
500uL/EP pipe 1X Binding solution is resuspended；
2) attached cell in 6 orifice plates is washed 2 times with cold PBS, is digested with the pancreatin (hole 500uL/) without EDTA thin
Born of the same parents；
3) after cell dissociation gets off, the hole PBS 1ml/ is added, cell suspension is transferred to the 1.5mlEP pipe marked,
Supernatant is abandoned in room temperature 1000rpm 3min centrifugation, and PBS resuspension washed once, then room temperature 1000rpm 3min centrifugation.Supernatant is abandoned, is added
Enter 1X Annexin V Binding Buffer to wash 1 time, room temperature 1000rpm 3min centrifugation, 500uL/EP pipe 1X
Binding solution is resuspended；
4) by the supernatant being resuspended cell and the corresponding group of attached cell mix, take 200uL cell mixing liquid to turn
The new 1.5mlEP pipe marked is moved to, 5uL/ pipe Annexin V is added, room temperature, which is protected from light, is incubated for 15min；
5) 1X Annexin V Binding Buffer washing, room temperature 1000rpm 3min centrifugation, 200uL 1X is added
Annexin V Binding Buffer is resuspended, and 5uL/ pipe PI is added, and room temperature, which is protected from light, is incubated for 15min；
6) 1X Annexin V Binding Buffer washing, room temperature 1000rpm 3min centrifugation, 200uL 1X is added
Annexin V Binding Buffer is resuspended；
7) 96 orifice plates, upper machine testing in 4h are transferred to.
As a result as shown in figure each in Fig. 4, erucic acid intervention group energy significant effective reduces cell caused by influenza virus and withers in early days
Die the cell number with late apoptic.
Six erucic acid of embodiment can inhibit the activation of Apoptosis precursor molecule pro-caspase3 caused by influenza virus with
And the consumption of substrate PARP
1, A549 cell kind is long to single layer in 6 orifice plates, discards former culture medium, PBS is added and washes twice, then plus containing influenza
Serum-free DMEM/DF12 (1:1) the culture medium adherent cell 2h of viral A/PR8/3/4 (H1N1) (MOI=0.1).It is washed with PBS
Cell removes unadsorbed extra virus.If control group (for normal A549 cell), viral group, pharmaceutical intervention group, drug alone
Group；Wherein, pharmaceutical intervention group uses mode of drug, and erucic acid (100 μ g/ml, 200 μ of various concentration are added in pharmaceutical intervention group
g/ml,300μg/ml)；Viral group is not added with pharmaceutical intervention.Drug alone group, A549 cell are added 200 without viruses adsorption
The erucic acid of μ g/ml.Continue 37 DEG C to cultivate 24 hours.
2, it then, extracts total protein of cell and carries out western blot (the corresponding step of detailed process reference embodiment three
It is rapid to carry out), use rabbit-anti people primary antibody pro-caspase3 antibody (being purchased from GeneTex company, GTX110543) detection pro-
Caspase3 molecule uses rabbit-anti people primary antibody PARP antibody (being purchased from GeneTex company, GTX100573) detection PARP molecule.Sheep
Anti-rabbit secondary antibody is purchased from U.S. Earthox company.
As a result as shown in figure 5, influenza A/PR8/3/4 (H1N1) infection A549 can obviously make apoptosis precursor molecule after for 24 hours
Pro-caspase3 activation is activist caspase3, so that the amount of precursor molecule pro-caspase3 is reduced, erucic acid can have
Effect inhibits the activation and reduction of Apoptosis precursor molecule pro-caspase3 caused by influenza virus, and erucic acid intervention group
The consumption of the substrate PARP of caspase3 is also reduced also confirms that erucic acid can inhibit cell caused by influenza virus to wither from another point of view
It dies.
The protective effect of the internal erucic acid influenza virus infected of embodiment seven
1) by week old 4-6 weeks, female, weight 14-16g, purchased from Guangdong Medical Lab Animal Center (credit number:
SYXK (Guangdong) 2013-0002) SPF grades of BABL/C mouse is randomly divided into five groups, it is respectively as follows: Control group (normal group or PBS
Group), viral group (IAV group or virus group), viral group (IAV group or virus group)+Oseltamivir group (60mg/kg/day), and
Erucic acid intervention group: viral group of (IAV group or virus group)+erucic acid (50mg/kg/day), viral group (IAV group or virus group)+mustard
Sour (100mg/kg/day)；
2) by after the lung adapted strain freeze thawing of influenza virus A/PR8/3/4 (H1N1) mouse, it is diluted to 1LD50, mouse is light through ether
After degree anesthesia, influenza infection model is prepared through 50 μ l of collunarium；Control group instills the PBS of equivalent with method；
3) 2 days before preparing BABL/C mouse influenza virus infection model, erucic acid intervention group is given by stomach-filling mode respectively
Give erucic acid (given low: 50mg/kg/day；100mg/kg/day), continuous 7 days, 1 time a day, stop giving within the 5th day after infection
Medicine.Oseltamivir drug control group then gives Oseltamivir 60mg/kg/day, other operations of the group are the same as erucic acid intervention group.It is right
Same amount of normal saline is then given once daily simultaneously according to group and virus group；
4) influenza infection model is prepared the 5th day, eyeball takes blood to put to death mouse, mouse lung lesion is dissected and observed, and weigh
Weight and lung weight measure Lung Exponent (Lung Exponent=lung weight (g)/weight (g) × 100), evaluate the variation of mouse Lung Exponent；
5) alveolar wass is carried out simultaneously, uses total protein concentration in BCA determination of protein concentration kit detection irrigating solution；
6) it takes mouse lung to save rapidly to liquid nitrogen simultaneously, surveys inflammatory factor IL-6 with liquid-phase chip technology after lung homogenate, resist
The concentration of scorching factor IL-10.
7) the 15th day after remaining ob mouse to virus infection, the death time of every mouse is recorded, calculates last put down
Equal time-to-live and survivorship curve.
As a result as shown in each figure of A-H in Fig. 6.Wherein, in Fig. 6, A is that erucic acid influences the Lung Exponent of virus infection mouse, A
It has been shown that, in terms of influenza model preparation, virus group Lung Exponent is within the scope of 1.5-2.0, Oseltamivir drug control group Lung Exponent
Within the scope of 0.5-1.0, have compared with viral group significant difference (p < 0.01), mouse influenza models is prompted to be successfully prepared.Mustard
The Lung Exponent of sour intervention group has significant statistical difference (p < 0.01) compared with virus group.In Fig. 6, B is erucic acid to virus infection
The adjustment effect of protein concentration, B are shown in mouse bronchoalveolar lavage fluid, and protein content significantly increases in virus group bronchoalveolar lavage fluid.
Protein content is substantially less than viral group in erucic acid intervention group mouse bronchoalveolar lavage fluid.Erucic acid is prompted to intervene obvious inhibition influenza cause small
Mouse lung is seriously damaged.In Fig. 6, C is adjustment effect of the erucic acid to IL-6 in the homogenate of virus infection mouse lung tissue, and D is erucic acid
To the adjustment effect of TNF-α in the homogenate of virus infection mouse lung tissue, E is adjustment effect of the erucic acid to MCP-1, and F is erucic acid pair
The adjustment effect of MIP-1, G are erucic acid to the adjustment effect of GM-CSF, and C-G is shown, prepares influenza infection model the 5th day, virus
Inflammatory factor IL-6 in group mouse lung tissue homogenate is significantly increased, and erucic acid intervention group obviously inhibits the inflammation in lung homogenate
The level of factor IL-6, and anti-inflammatory factors TNF-α, MCP-1, MIP-1, GM-CSF level increase.In Fig. 6, H shows erucic acid pair
The effect of virus infection mouse, as seen from the figure, compared with viral group, interstitial lung narrows for erucic acid treatment group, and inflammatory cell infiltration subtracts
It is few.The mean survival time of erucic acid intervention group mouse has significant statistical difference obviously than viral group leader.
The experiment of above-mentioned inside and outside sufficiently shows erucic acid to the duplication of influenza a virus infection restrovirus itself, mediation
Inflammatory reaction and Apoptosis have obvious inhibitory activity, and the possible mechanism of action is that antiviral targets be both directed to virus also for place
It is main, secondary excessive inflammation and Apoptosis after inhibiting progeny virus also to adjust host infection virus while generation.Therefore,
Erucic acid can be applied to prepare novel Tamiflu.Compared with existing drug, novel Tamiflu is prepared using erucic acid and is had
Standby outstanding advantage: 1, erucic acid antiviral targets be both directed to virus also for host, worked along both lines, significant in efficacy；2, erucic acid is without bright
Aobvious toxic side effect；3, erucic acid is plant monomer ingredient, and structure is clear, and chemical property is stablized, and is easy to quality control；4, erucic acid source
It is abundant, it is cheap and easy to get, mass production demand can be met.Erucic acid can combine individually or with the substance of other medicinal licenses, easily make
At the various peroral dosage forms such as tablet, hard capsule, soft capsule, granule, dripping pill.
It is those of ordinary skill in the art according to it if the specific experiment operation that embodiment is related in text is not specified
The common knowledge or conventional technical means of grasp can be appreciated or know, no longer repeat one by one this.
The above described is only a preferred embodiment of the present invention, limitation in any form not is done to the present invention, therefore
All contents without departing from technical solution of the present invention, it is made to the above embodiment according to the technical essence of the invention any simply to repair
Change, equivalent variations and modification, all of which are still within the scope of the technical scheme of the invention.
Claims (5)
1. application of the erucic acid in the drug of preparation prevention or treatment Flu-A.
2. application according to claim 1, which is characterized in that the erucic acid has the following structure formula:
3. application according to claim 1, which is characterized in that the erucic acid preparation for inhibit influenza A virus and
It is applied in the drug of its inflammatory reaction mediated.
4. application according to claim 1, which is characterized in that the drug contains pharmaceutically acceptable carrier or auxiliary
Material.
5. application according to claim 1, which is characterized in that pharmaceutically acceptable dosage form is made in the drug.
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| CN101921823A (en) * | 2010-05-05 | 2010-12-22 | 中国科学院生物物理研究所 | Method for screening SARS corona virus major protease inhibitor from traditional Chinese medicine and screened SARS corona virus major protease inhibitor |
| CN105769877A (en) * | 2016-03-10 | 2016-07-20 | 广州医科大学附属第医院 | Application of beta-sitosterol to preparation of medicine for treating or preventing influenza A |
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| CN101921823A (en) * | 2010-05-05 | 2010-12-22 | 中国科学院生物物理研究所 | Method for screening SARS corona virus major protease inhibitor from traditional Chinese medicine and screened SARS corona virus major protease inhibitor |
| CN105769877A (en) * | 2016-03-10 | 2016-07-20 | 广州医科大学附属第医院 | Application of beta-sitosterol to preparation of medicine for treating or preventing influenza A |
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