CN108014101A

CN108014101A - Application of the erucic acid in the medicine for preparing prevention or treatment Flu-A

Info

Publication number: CN108014101A
Application number: CN201810045581.9A
Authority: CN
Inventors: 杨子峰; 李菁; 梁晓丽; 周倍贤; 潘锡平; 钟南山
Original assignee: STATE KEY LABORATORY OF RESPIRATORY DISEASE; Guangzhou Institute Of Respiratory Health; First Affiliated Hospital of Guangzhou Medical University
Current assignee: STATE KEY LABORATORY OF RESPIRATORY DISEASE; Guangzhou Institute Of Respiratory Health; First Affiliated Hospital of Guangzhou Medical University
Priority date: 2018-01-17
Filing date: 2018-01-17
Publication date: 2018-05-11
Anticipated expiration: 2038-01-17
Also published as: CN108014101B

Abstract

The present invention provides new application of the erucic acid in terms of preparation is used to prevent or treat Flu-A medicine.The present invention also provides a kind of medicine for being used to preventing or treating Flu-A, the active ingredient of the medicine includes erucic acid.Present inventor has found that the inflammatory reaction that erucic acid replicates influenza A virus and mediates has significant inhibitory activity.Confirmed using liquid-phase chip technology, erucic acid can substantially suppress the unconventionality expression of the H1N1 virus infection A549 cellular inflammation factors and be in dose-dependence；Confirmed using Western blot, erucic acid can inhibit and the activation of host inflammation associated signal paths and be in dose-dependence；Confirmed using Annexin V FITC/PI double labellings flow cytometry, the Apoptosis of Host Cells that erucic acid can substantially suppress influenza H1N1 inductions is damaged and is in dose-dependence.Confirmed by zoopery, erucic acid can substantially suppress lung inflammation and the apoptosis damage of Influenza virus H1N1 induction, effectively extend the life span of mouse.

Description

Application of the erucic acid in the medicine for preparing prevention or treatment Flu-A

Technical field

The invention belongs to field of medicaments, is related to a kind of application of natural products in terms of Tamiflu is prepared, especially relates to And erucic acid is preparing the new application of prevention or treatment Flu-A medicine.

Background technology

Influenza (abbreviation influenza) is the acute respiratory disease as caused by influenza virus, has 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 Easily caused in youngster, the elderly or chronic in hospital and dead.The annual influenza in the whole world causes about 3,000,000 to 5,000,000 sternly Weight disease and about 250,000 to 500,000 death, the life and health of the serious threat 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 produced for antigenic drift and variation infects not for the production of vaccine Produce the protective effect of body.Therefore, influenza vaccines flu-prevention has certain hysteresis quality, and protective rate is also limited.Use at present Mainly there are two classes in the chemicals for the treatment of of influenza：Adamantane amine (amantadine and Rimantadine) and neuraminidase suppress Agent (Oseltamivir and zanamivir), usually need to disease early stage (symptom occur after 48 it is small when it is interior) administration, and due to act on target Point is single to have there is drug resistance, significantly impacts curative effect.It is therefore, clinical that there is an urgent need for 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 suppress the virus of invasion.The inflammatory reaction of appropriateness is conducive to the clear of influenza virus Remove.But excessive inflammatory response can cause " inflammatory factor storm ", cause the serious tissue damage of body, this is that influenza causes death One of the main reason for.Therefore, using suppress influenza virus replicate and excessive inflammatory response as target medicament research and development become grind Study carefully hot spot.Also modulate host inherent immunity reacts relevant signal path and inflammatory factor while such medicine is directly antiviral Secretion, it is therefore evident in efficacy and be not easy drug resistance.

Erucic acid (Erucic acid) is widespread in nature, and mustard is refining to obtain 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；Acidifying 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 on erucic acid in suppressing virus replication and Effect in terms of the inflammatory reaction of suppression influenza A virus mediation did not appeared in the newspapers.

The content of the invention

It is an object of the invention to provide new application of the erucic acid in terms of preparation is used to prevent or treat Flu-A medicine.

For this reason, the application the present invention provides erucic acid in the medicine for preparing prevention or treatment Flu-A.

The erucic acid has following structural formula：

The present invention also provides erucic acid to prepare the medicine for the inflammatory reaction for being used to suppress influenza A virus duplication and its mediation Application in thing.

Present invention also offers a kind of medicine for being used to preventing or treating Flu-A, the active ingredient of the medicine includes mustard Acid, erucic acid can be used as sole active agent, can also be combined with the material of other medicinal licenses.The medicine can further comprise pharmacy Upper acceptable carrier or auxiliary material, can be prepared into pharmaceutically acceptable formulation, formulation 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, it 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, glycerine, gather Ethylene glycol, ethanol, propyl alcohol, starch slurry, dextrin, syrup, honey, glucose solution, mucialga of arabic gummy, gelatine size, carboxymethyl are fine The plain sodium of dimension, lac, methylcellulose, potassium phosphate, polyvinylpyrrolidone etc.；Pharmaceutically common disintegrant, such as dry shallow lake Powder, alginate, agar powder, laminaran, sodium acid carbonate 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..No longer enumerated on pharmaceutically acceptable carrier or auxiliary material, those of ordinary skill in the art can be according to institute The common knowledge of grasp carries out specifically chosen.

Present invention also offers a kind of medicine for the inflammatory reaction for being used to suppress influenza A virus and its mediation, the medicine Active ingredient include erucic acid, erucic acid can be used as sole active agent, can also be combined with the material of other medicinal licenses.The medicine It can further comprise pharmaceutically acceptable carrier or auxiliary material, pharmaceutically acceptable formulation, formulation can be prepared into as needed The for example, various peroral dosage forms such as tablet, hard shell capsules, 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, glycerine, polyethylene glycol, ethanol, propyl alcohol, starch slurry, dextrin, syrup, honey, glucose solution, mucialga of arabic gummy, Gelatine size, sodium carboxymethylcellulose, lac, methylcellulose, potassium phosphate, polyvinylpyrrolidone etc.；It is pharmaceutically common to collapse Solve agent, for example, dry starch, alginate, agar powder, laminaran, sodium acid carbonate 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..No longer enumerated on pharmaceutically acceptable carrier or auxiliary material, this area is common Technical staff can carry out specifically chosen according to the common knowledge grasped.Present inventor has found erucic acid to influenza A virus Replicate and its inflammatory reaction of mediation has significant inhibitory activity.Present inventor is using mdck cell lesion method, supernatant Progeny virus Inhibition test confirms that erucic acid can substantially suppress the self-replication of H1N1 viruses；Confirmed using Suspension array technique, Erucic acid can substantially suppress inflammatory factor IL-1beta, IL-6, IL-21, IP-10, IL-17A, IL- of influenza virus induction 17F, IFN-γ, TNF-α unconventionality expression and be in dose-dependence；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, in dose-dependence. Confirm that erucic acid can substantially suppress Influenza virus H1N1 and 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 medicine, prepare anti influenza newtype drug using erucic acid and possess outstanding advantage：1st, erucic acid action target Both for virus, also for host cell, therefore it is not likely to produce drug resistance；2nd, erucic acid is without obvious toxic-side effects；3rd, erucic acid is plant list Body component, structure is clear and definite, and chemical property is stablized, and is easy to quality control；4th, erucic acid abundance, it is cheap and easy to get, it can meet big life Production demand.

Brief description of the drawings

Fig. 1 shows inhibitory action of the erucic acid to 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 using Bio-plex Suspension array techniques in the infection induced place of protein level detection erucic acid infected by influenza The effect of chief cell inflammatory response.

Fig. 4 shows the Apoptosis of Host Cells damage that Flow cytometry erucic acid induces influenza A/PR8/3/4 (H1N1) Inhibitory action.

Fig. 5 shows in the case of virus infects A549 cells, using western blot technology for detection erucic acid Apoptosis 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 is：Erucic acid is to infection disease The Lung Exponent of malicious mouse influences；B is：Adjustment effect of the erucic acid to protein concentration in virus infection mouse bronchoalveolar lavage fluid；C is： The adjustment effect of IL-6 during erucic acid is homogenized virus infection mouse lung tissue；D is：Erucic acid is homogenized virus infection mouse lung tissue The adjustment effect of middle TNF-α；E is：Adjustment effect of the erucic acid to MCP-1；F is：Adjustment effect of the erucic acid to MIP-1；G is：Mustard Adjustment effect of the acid to GM-CSF；H is：Effect of the erucic acid to virus infection mouse.

Embodiment

Technical scheme is described further below in conjunction with the accompanying drawings.If instrument or reagent are not in embodiment Special instruction, it is using this area conventional reagent or instrument, the conventional products obtained can be bought by market.

One erucic acid of embodiment causes common influenza virus the inhibitory action of cytopathy

1st, material：Cell and virus

The mdck cell (being less than for 25 generations) of low algebraically 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.

2nd, reagent：

DMEM/DF12(1：1) culture medium is purchased from Gibco companies；MTT is purchased from sigma companies, and 5mg/mL MTT solution= The MTT+100mL DMEM/DF12 (1 of 50mg：1) culture medium；Virus-culturing fluid (the TPCK pancreatin of the 1.5ug/mL containing final concentration)： 100mL DMEM/DF12(1：1) the 1mg/mLTPCK pancreatin of nutrient 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 with clinical, 2011,26 (5)：381-383] carry out, It can be bought by market or be obtained using existing other methods.

3rd, inhibitory action 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, 100 μ L/ holes, when 37 DEG C of incubations 2 are small are added；

3) virus incubation liquid is discarded, pharmaceutical intervention group adds the Virus culture of the TPCK pancreatin with the 1.5ug/mL containing final concentration 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).Result is observed after when 37 DEG C of cultures 48 are small；

4) observation caliber such as following table

6 grades of standards of cytopathy (CPE) caused by 1 virus of table

5) IC of CPE methods₅₀Formula

Drug effect inhibiting rate=1- of CPE is average per hole extent of disease (%)

CPE distances than=(>50% inhibiting rate -50%)/(>50% inhibiting rate-<50% inhibiting rate)

The IC of CPE methods₅₀=less than the drug concentration X2 of 50% inhibiting rate^{Distance than}

As shown in the following Table 2, as a result illustrate that erucic acid can effectively suppress influenza virus A/PR/8/34 (H1N1), A/GZ/ Cytopathy caused by GIRD07/09 (H1N1), A/HK/8/68 (H3N2).

Table 2

Inhibitory action of two erucic acid of embodiment to influenza A/PR8/3/4 (H1N1) cells and supernatant generation of neutrons viruses

1st, material：Cell and virus

A549 cells are purchased from ATCC, are stored in this laboratory

2nd, inhibitory action of the erucic acid to influenza A/PR8/3/4 (H1N1) progeny virus

1) 6 orifice plate individual layer A549 cells discard former culture medium, add PBS and wash twice；

2) the serum-free DMEM/DF12 (1 containing influenza virus A/PR8/3/4 (H1N1) is added：1) culture medium, 37 DEG C of absorption Cell 2h；

3) cell is washed with PBS to remove unadsorbed virus；

4) mode of drug is used, pharmaceutical intervention group adds erucic acid (100 μ g/ml, 200 μ g/ml, 300 of various concentrations μ g/ml), drug alone group adds the erucic acid of 200 μ g/ml, and the A549 cells of drug alone group are adsorbed without influenza virus, 37 DEG C Cultivate 24 it is small when；

5) cells and supernatant is collected；

6) 96 orifice plate MDCK cell monolayers are washed 1 time with PBS；

7) the 100 μ L/ holes of cells and supernatant collected above are added, when 37 DEG C of incubations 2 are small；

8) 96 orifice plates sop up supernatant, add the 100 μ L/ holes of virus-culturing fluid of the TPCK pancreatin of the 1.5ug/mL containing final concentration, When 37 DEG C of cultures 48 are small；

9) observation is as a result, immediately arrive at TCID₅₀。

The results are shown in Figure 1, and erucic acid can effectively suppress influenza virus A/PR/8/34 (H1N1) cells and supernatant generation of neutrons The titre of virus.

Three erucic acid infected by influenza A/PR8/3/4 (H1N1) of embodiment induces the effect of host signal path

1st, 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 are public purchased from Ameresco Department；Dodecyl sodium sulfate (SDS) is purchased from sigma companies of the U.S.；Ammonium Persulfate 98.5 (APS) is purchased from Ameresco companies of the U.S.； TEMED (N,N methylene bis acrylamide) is purchased from Bio-rad companies of the U.S.；RIPA lysates are purchased from Thermo companies of the U.S.； PMSF is purchased from sigma companies of the U.S.；Cocktail (protease inhibitors) is purchased from sigma companies of the U.S.；BCA determination of protein concentration Kit is purchased from Thermo companies of the U.S.；Easysee Western marker (20-90kDa) are purchased from Chinese T ransgen Biotech companies；Blue plus II protein marker are purchased from Chinese T ransgen Biotech companies；Pvdf membrane is purchased From Life science companies of the U.S.；Skimmed milk power is purchased from Gibco companies；Hyclone is purchased from Gibco companies；Rabbit-anti people p- P65 antibody is purchased from CST companies of the U.S.；Rabbit-anti people p65 antibody is purchased from CST companies of the U.S.；Rabbit-anti people p-p38 antibody is purchased from the U.S. CST companies；Rabbit-anti people p38 antibody is purchased from CST companies of the U.S.；

Rabbit-anti people p-pERK antibody is purchased from CST companies of the U.S.；Rabbit-anti people ERK antibody is purchased from CST companies of the U.S.；Rabbit-anti people p- JNK antibody is purchased from CST companies of the U.S.；Rabbit-anti people JNK antibody is purchased from CST companies of the U.S.；Rabbit-anti people p-AKT antibody is purchased from the U.S. CST companies；Rabbit-anti people AKT antibody is purchased from CST companies of the U.S.；Rabbit-anti people GAPDH antibody is purchased from CST companies of the U.S.；Goat-anti rabbit two It is anti-to be purchased from Earthox companies of the U.S..

2nd, erucic acid suppresses the activation of influenza inducing host cell signal path

1) cell is laid on 6 orifice plates by the density for adjusting A549 cells, is positioned in incubator, after cultivating 12h cell attachments It is stand-by；

2) setting of experimental group is carried out, including：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 cells are taken out, discards culture medium, added PBS and wash twice, then added and contain influenza virus A/PR8/ The DMEM/DF12 (1 of 3/4 (H1N1) (MOI=0.1) serum-free：1) culture medium 1ml/ holes adherent cell 2h；

4) unadsorbed unnecessary virus is washed away with PBS, pharmaceutical intervention group adds erucic acid (100 μ g/ml, 200 of various concentrations μ g/ml, 300 μ g/ml), drug alone group adds the erucic acid of 200 μ g/ml.37 DEG C continue culture 24 it is small when；

5) 6 orifice plate of cell is placed on ice, discards culture supernatant, and washed twice with cold PBS；Washing finishes, rapidly Add 130ul cell pyrolysis liquids RIPA (containing protease inhibitors cocktail, itself and cell pyrolysis liquid RIPA volume ratios (V/ V it is) 1：100 additions and the PMSF of final concentration of 10uM), carry out extraction total protein of cell；

6) 1.5mlEP pipes are marked and are placed in that cell lysate on ice, is transferred to each group EP pipes marked, and 30min is shaken on shaking table, allows albumen fully to crack；

7) cell lysate is moved to the centrifuge of 4 DEG C of precoolings, carries out 13000rpm × 15min centrifugations；

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 kits specification, so BCA detection working solution 200ul/ hole (configuration work liquid A are hole-specifically added afterwards:B=1：50), 37 DEG C of incubation 30min after mixing, take out 96 orifice plates are cooled to room temperature, and OD values are measured in absorbance 562nm in microplate reader.Standard curve is drawn, draws protein concentration.

10) preparation of sds page：

According to recipe configuration 10%SDS polyacrylamide gel electrophoresis separation gels：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) + 0.004ml TEMED, quickly irrigate separation gel between separation of glasses plate after fully mixing, perfusion finishes adds ddH20 on upper strata Separation glue liquid surface is sealed, waits 30min separation gels to condense intact；It is dense according to recipe configuration 5%SDS polyacrylamide gel electrophoresises 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, quickly irrigate dense after fully mixing between glass plate Contracting glue, irrigates and finishes insertion comb, and excludes bubble, waits 30min concentration gelling knots intact.

11) it is loaded：

1 is pressed with 5 times of concentration SDS polyacrylate hydrogels sample loading buffers (5Xloading buffer) and sample (20 μ g)：4 ratios Example mixing, 95 DEG C of denaturation 10min, being immediately passed to cooled on ice 10min prevents protein renaturation.3000rpm × 1min is centrifuged, hole-specifically Sample-adding；

12) electrophoresis：

With Bio-Rad vertical electrophoresis apparatus, 80V constant pressures electrophoresis about (1.5-2) h, treats that bromophenol blue indicator reaches separation gel bottom Stop electrophoresis during portion；

13) protein transferring film：

Pvdf membrane first soaks 15min with methanol.Gel is removed after electrophoresis, unnecessary Blank gel is cut off, after trimming 6 Whatman3M filter paper are cut by its size.Neat postposition is stacked by the order of 3 filter paper-gel-filter paper of pvdf membrane -3 Turn in wet on negative plate, with the bubble between exhaust bubble plate venting gel and pvdf membrane, in freezer, turned with the constant current of 380mA Film 2h.After transferring film, take 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 companies, ERK1/2MAPK, P-JNK, The corresponding dilution proportion antibody of P-AKT specifications, is added to and has closed on the pvdf membrane finished, be placed in Cool Room 4 DEG C and be incubated Night；

15) second day, take out pvdf membrane and recover to room temperature, remove primary antibody and wash film 3 times, 5min/ times with washing lotion TBST. Then add (V/V) 1 by volume:The 1000 goat-anti rabbit secondary antibodies with horseradish peroxidase (HRP) coupling being diluted, room Temperature is incubated 1h.Remove secondary antibody and wash film 3 times, 5min/ times with washing lotion TBST；

16) film is taken out, drip-dry moisture on filter paper, adds ECL immunofluorescences and shine color developing agent；

17) tabletting is carried out in darkroom, and film is fixed in developing liquid developing and fixing solution；

18) interpretation of result is with arranging.

As a result after arranging as shown in Fig. 2, after influenza A/PR8/3/4 (H1N1) infection A549 cells 24h, cell-signaling pathways NF-B, P38MAPK, ERK1/2MAPK, P-JNK, P-AKT are substantially activated.Erucic acid substantially suppresses influenza A/PR8/3/4 (H1N1) and lures 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

1st, 6 orifice plate individual layer A549 cells, discard archaeocyte culture medium, add PBS and wash twice, and then add containing influenza disease The DMEM/DF12 (1 of malicious A/PR8/3/4 (H1N1) serum-free：1) culture medium adherent cell.After 2h, cell is washed with PBS and is removed Go unadsorbed virus.If control group (for normal A549 cells), viral group, virus+pharmaceutical intervention group, drug alone group；Its In, virus+pharmaceutical intervention group uses mode of drug, and pharmaceutical intervention group adds erucic acid (100 μ g/ml, 200 μ of various concentrations g/ml、300μg/ml)；Viral group is not added with pharmaceutical intervention.Drug alone group, its A549 cell add 200 without viruses adsorption The erucic acid of μ g/ml.Continue culture 24 it is small when after, collect cells and supernatant in 1.5mlEP manage.Then, be placed in 4 DEG C of precoolings 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.

2nd, cytokine content in Bio-plex Suspension array techniques detection cell conditioned medium：

1) take out sample to be tested slowly to be thawed on ice, take out Bio-Plex suspension chip inspecting reagent units (Bio Rad companies), make it restore to room temperature；

2) position of standard sample wells, blank well and sample well on experiment flat underside, standard sample wells and each 2 of blank well are set Multiple holes；

3) (Bio-Plex suspensions chip inspecting reagent unit) ratio adds detection buffer solution assay to specifications Buffer is diluted antibody microballoon, and abundant vortex homogeneous magnetic microballoon, is added in flat underside per hole 50ul microballoon mixed liquors；

4) 100ul dcq buffer liquid wash buffer are added per 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 are added separately to standard sample wells, the sample of flat underside In sample wells, blank well adds assay buffer, with sealed membrane sealing plate；

6) it is incubated：Lucifuge is wrapped with aluminium foil, flat underside is incubated at room temperature 30min in 96 plate shaker 300rpm；

7) wash：Incubation, which finishes flat underside being placed on magnetic frame, discards liquid.100ul wash are added per hole Buffer, and flat underside is placed on magnetic frame and discards liquid, repeat this process and wash 3 times；

8) detection antibody is added：Detection antibody is shaken up using preceding be fully vortexed, ratio adds to specifications by detection antibody 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, 25ul detection antibody is added per hole in flat underside.At room temperature, flat underside is placed in shaking table 300rpm is incubated 30min；

9) wash：Incubation, which finishes flat underside being placed on magnetic frame, discards liquid, and 100ul wash are added per 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 fluoresceins PE marks is added per hole in flat underside, room temperature is in 96 plate shakers 300rpm is incubated 10min；

11) wash:Incubation, which finishes flat underside being placed on magnetic frame, discards liquid, and 100ul wash are added per hole Buffer, is placed on magnetic frame and discards wash buffer, 3 detections of repeated washing；

12) flat underside is added to 125ul detections buffer solution assay buffer, lucifuge rocker 1100rpm* at room temperature per hole 30 seconds, it is detected with Bio-plex 200；

As a result as shown in each figure in Fig. 3, erucic acid obvious inflammatory factor (IL- for suppressing influenza virus induction after intervening 1beta, IL-6, IL-21, IP-10, IL-17A, IL-17F, IFN-γ, TNF-α) level.Prompting erucic acid may pass through suppression The effect of influenza infection is treated in the excessive inflammation response of influenza infection induction body to play.

Embodiment five is using Annexin V-FITC/PI double labelling Flow cytometry erucic acid to influenza A/PR8/3/4 (H1N1) inhibitory action of the Apoptosis of Host Cells damage of (MOI=0.1) induction

1st, A549 cells kind discards former culture medium, adds PBS and wash twice, then plus contain influenza in 6 orifice plate length to individual layer The serum-free DMEM/DF12 (1 of viral A/PR8/3/4 (H1N1) (MOI=0.1)：1) culture medium adherent cell 2h.Washed with PBS Cell removes unadsorbed unnecessary virus.If control group (for normal A549 cells), viral group, pharmaceutical intervention group, drug alone Group；Wherein, pharmaceutical intervention group uses mode of drug, and pharmaceutical intervention group adds erucic acid (100 μ g/ml, 200 μ of various concentrations g/ml、300μg/ml)；Viral group is not added with pharmaceutical intervention.Drug alone group, its A549 cell add 200 without viruses adsorption The erucic acid of μ g/ml.When 37 DEG C of cultures 24 of continuation are small.

2nd, the cell collected in 6 orifice plates carries out streaming Annexin V (being purchased from affymetrix eBioScience companies) 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, are then added 1X Annexin V Binding Buffer and are washed 1 time, room temperature 1000rpm 3min centrifugations, 500uL/EP pipe 1X Binding solution are resuspended；

2) attached cell on 6 orifice plates is washed 2 times with cold PBS, is digested with the pancreatin (500uL/ holes) without EDTA thin Born of the same parents；

3) after cell dissociation gets off, PBS 1ml/ holes are added, cell suspension is transferred to the 1.5mlEP pipes marked, Room temperature 1000rpm 3min are centrifuged, and abandon supernatant, and PBS is resuspended and washed once, then room temperature 1000rpm 3min centrifugations.Supernatant is abandoned, is added Enter 1X Annexin V Binding Buffer to wash 1 time, room temperature 1000rpm 3min centrifugations, 500uL/EP pipes 1X Binding solution are resuspended；

4) cell in the supernatant being resuspended and the corresponding group of attached cell are mixed, takes 200uL cells mixing liquid to turn The new 1.5mlEP marked pipes are moved to, add 5uL/ pipe Annexin V, room temperature lucifuge is incubated 15min；

5) 1X Annexin V Binding Buffer washings, room temperature 1000rpm 3min centrifugations, 200uL 1X are added Annexin V Binding Buffer are resuspended, and add 5uL/ pipe PI, and room temperature lucifuge is incubated 15min；

6) 1X Annexin V Binding Buffer washings, room temperature 1000rpm 3min centrifugations, 200uL 1X are added Annexin V Binding Buffer are resuspended；

7) 96 orifice plates are transferred to, machine testing is gone up in 4h.

As a result as shown in each figure 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 suppress the activation of Apoptosis precursor molecule pro-caspase3 caused by influenza virus with And the consumption of substrate PARP

2nd, then, carrying out extraction total protein of cell progress western blot, (detailed process is accordingly walked with reference to embodiment three It is rapid to carry out), detect pro- using rabbit-anti people primary antibody pro-caspase3 antibody (being purchased from GeneTex companies, GTX110543) Caspase3 molecules, PARP molecules are detected using rabbit-anti people primary antibody PARP antibody (being purchased from GeneTex companies, GTX100573).Sheep Anti-rabbit secondary antibody is purchased from Earthox companies of the U.S..

The results are shown in Figure 5, can substantially make apoptosis precursor molecule after influenza A/PR8/3/4 (H1N1) infection A549 24h Pro-caspase3 activation is activist caspase3, so that the amount of precursor molecule pro-caspase3 is reduced, erucic acid can have Effect suppresses 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 suppress cell caused by influenza virus and wither from another point of view Die.

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's (credit number： SYXK (Guangdong) 2013-0002) SPF grades of BABL/C mouse are randomly divided into five groups, be respectively：Control groups (normal group or PBS Group), viral group (IAV groups or virus groups), viral group (IAV groups or virus groups)+Oseltamivir group (60mg/kg/day), and Erucic acid intervention group：Viral group of (IAV groups or virus groups)+erucic acid (50mg/kg/day), viral group (IAV groups or virus groups)+mustard Sour (100mg/kg/day)；

2) by after the lung adapted strain freeze thawing of influenza virus A/PR8/3/4 (H1N1) mouse, 1LD50 is diluted to, mouse is light through ether After degree anesthesia, influenza infection model is prepared through 50 μ l of collunarium；Control groups instill the PBS of equivalent with method；

3) 2 days before BABL/C mouse influenza virus infection models are prepared, erucic acid intervention group is given by gavage mode respectively Give erucic acid (given low：50mg/kg/day；100mg/kg/day), continuous 7 days, 1 time a day, stop within the 5th day after infection to 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 Normal saline then is given once daily at the same time according to group and virus group；

4) influenza infection model is prepared the 5th day, eyeball takes blood to put to death mouse, anatomic observation mouse lung lesion, and weighs Weight and lung weight, measure Lung Exponent (Lung Exponent=lung weight (g)/weight (g) × 100), evaluates the change of mouse Lung Exponent；

5) alveolar wass is carried out at the same time, total protein concentration in irrigating solution is detected using BCA determination of protein concentration kit；

6) while take mouse lung to preserve to liquid nitrogen rapidly, inflammatory factor IL-6 is surveyed with liquid-phase chip technology after lung homogenate, resist The concentration of scorching factor IL-10.

7) the 15th day after remaining ob mouse is infected to virus, 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 figures 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 in the range of 1.5-2.0, Oseltamivir drug control group Lung Exponent In the range of 0.5-1.0, there is significant difference (p compared with viral group<0.01), prompting mouse influenza models are successfully prepared.Mustard The Lung Exponent of sour intervention group has notable significant difference (p compared with virus is organized<0.01).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 raises 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 suppression influenza cause small Mouse lung major injury.In Fig. 6, C is the adjustment effect of IL-6 during erucic acid is homogenized virus infection mouse lung tissue, and D is erucic acid To virus infection mouse lung tissue be homogenized in TNF-α adjustment effect, E for erucic acid to the adjustment effect of MCP-1, 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 show, prepares influenza infection model the 5th day, viral Inflammatory factor IL-6 in group mouse lung tissue homogenate is significantly raised, and erucic acid intervention group substantially suppresses the inflammation in lung homogenate The level of factor IL-6, and anti-inflammatory factors TNF-α, the horizontal rise of MCP-1, MIP-1, GM-CSF.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 notable significant difference substantially than viral group leader.

The experiment of above-mentioned inside and outside fully shows, duplication of the erucic acid to influenza a virus infection restrovirus itself, mediate Inflammatory reaction and Apoptosis have obvious inhibitory activity, and the possible mechanism of action is antiviral targets both for virus also for place It is main, suppress also to adjust secondary excessive inflammation after host infection virus and Apoptosis while progeny virus produces.Therefore, Erucic acid can be applied to prepare new Tamiflu.Compared with existing medicine, prepare new Tamiflu using erucic acid and have Standby outstanding advantage：1st, erucic acid antiviral targets are worked along both lines both for virus also for host, evident in efficacy；2nd, erucic acid is without bright Aobvious toxic side effect；3rd, erucic acid is plant monomer component, and structure is clear and definite, and chemical property is stablized, and is easy to quality control；4th, erucic acid source It is abundant, it is cheap and easy to get, it can meet big production requirement.Erucic acid can combine individually or with the material of other medicinal licenses, easily make The various peroral dosage forms such as piece agent, hard shell capsules, soft capsule, granule, dripping pill.

It is those of ordinary skill in the art according to it if the operation of embodiment is related in text specific experiment is not specified The common knowledge or conventional technical means of grasp can be appreciated or know, this is no longer repeated one by one.

The above described is only a preferred embodiment of the present invention, limitation in any form is not done to the present invention, therefore All contents without departing from technical solution of the present invention, the technical spirit according to the present invention any are simply repaiied to made for any of the above embodiments Change, equivalent variations and modification, in the range of still falling within technical solution of the present invention.

Claims

1. application of the erucic acid in the medicine for preparing prevention or treatment Flu-A.

2. application according to claim 1, it is characterised in that the erucic acid has following structural formula:

3. application according to claim 1, it is characterised in that the erucic acid prepare be used for suppress influenza A virus and Applied in the medicine of its inflammatory reaction mediated.

4. application according to claim 1, it is characterised in that the medicine contains pharmaceutically acceptable carrier or auxiliary Material.

5. application according to claim 1, it is characterised in that pharmaceutically acceptable formulation is made in the medicine.

6. a kind of medicine for being used to treat or prevent Flu-A, it is characterised in that the active ingredient of the medicine includes erucic acid.

7. medicine according to claim 6, it is characterised in that further include pharmaceutically acceptable carrier or auxiliary material.

8. a kind of medicine for the inflammatory reaction for being used to suppress influenza A virus and its mediation, it is characterised in that the medicine Active ingredient includes erucic acid.

9. medicine according to claim 8, it is characterised in that further include pharmaceutically acceptable carrier or auxiliary material.