CN106431960B - Application and its preparation of the Aurantiamide acetate in anti-influenza virus medicament - Google Patents
Application and its preparation of the Aurantiamide acetate in anti-influenza virus medicament Download PDFInfo
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- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
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Abstract
The present invention provides application of the Aurantiamide acetate in the drug for preparing resisiting influenza virus, for clinically the prevention and treatment of influenza provide a kind of effective drug now.A kind of method detaching Aurantiamide acetate from Radix Isatidis is also provided.Present inventor has found that Aurantiamide acetate has resisiting influenza virus effect, is in inhibiting effect to the cytopathic effect that influenza A virus mediates；Aurantiamide acetate action target is the signaling molecule of host cell rather than viral, can play antivirus action, can also play the effect for adjusting influenza induction excessive inflammation, be not likely to produce drug resistance.
Description
Technical field
The invention belongs to pharmaceutical technology fields, more particularly to Aurantiamide acetate answering in preparing anti-influenza virus medicament
With the method for further relating to detach Aurantiamide acetate from Radix Isatidis.
Background technology
Influenza virus belongs to the RNA virus of sub-thread minus strand, can cause acute and highly infectious breathing problem, seriously
Threaten the health of the mankind." the big influenza of Spain " of outburst in 1918 is Influenza epidemic situation the most serious in history, causes the whole world 5000
The death of more ten thousand people.The new epidemic situation of A/China/2013 (H7N9) viruses broken out in East China in March, 2013, by wide
General concern, until making a definite diagnosis 658 infection H7N9 cases in July, 2015, the death rate is up to 33.5%.It is applied to clinically prevention stream at present
Sense drug has two classes：M2 inhibitors of ion channels and neuraminidase inhibitor.But the PA due to being relied on when influenza gene replicates,
The RNA polymerase (RNA-dependent RNA polymerase, RDRP) that the RNA that PB1 and PB2 heterotrimers are constituted is relied on
Lack correction activity and, viral genome mutation rate is very high.Have a large amount of report two class drugs of influenza virus pair and generates drug resistance
Property.Therefore, the effective newtype drug utmost importance of prevention and control influenza pandemic is found.
Cause intracellular signal cascades reaction to be activated after influenza infection host cell, causes proinflammatory factor as interfered
The gene expressions such as element start host anti-virus immune response.But while influenza virus makes virus by manipulating host signal molecule
Itself is effectively replicated.Therefore, scholar both domestic and external proposes that it is fine to inhibit influenza duplication by targeting the signal path of host
Design Tamiflu R&D direction.
Nuclear factor kappaB (Nuclear factor-kB, NF-kB) is inducibility transcription factor.After NF-kB is activated, enter
Core is combined with the promoter of target gene, is regulated and controled numerous cell factors, chemokine, cell adhesion molecule, growth factor, is exempted from
The expression such as epidemic disease receptor adjust the physiological and pathologicals processes such as cell growth, differentiation, apoptosis, canceration.The activation of transcription factor NF-kB is
One of the outstanding feature of various pathogenic microorganism (including influenza virus) infection.After influenza infection, the NF-kB of activation enters core
By starting inflammatory factor, antiviral agent (IFN-α/β) and promoting expression of apoptotic gene so that host body is coped with and controlled in time
The further transmission of infection of continuation of influenza processed.The study found that NF-kB activation is inhibited to lead to antiviral agent IFN-β expression
Decline.Therefore, it is believed that NF-kB major functions start Immune responses of the antivirus.With this viewpoint on the contrary, it is other research shows that
Negativity is mutated molecules upstream IKK2 or IkB α, to inhibit NF-kB to activate, but finds that virus titer is decreased obviously.This research
As a result it is necessary to virus replication breeding to prompt our NF-kB.Further investigation revealed that its mechanism is viral activation NF-kB
And raise and promote antiapoptotic factors TRAIL or FasL expression and activate caspases active cell programmed deaths, to contribute to disease
Malicious nucleoprotein core output.It is another studies have shown that influenza virus activation NF-kB up-regulation cytokine signaling mortifier (SOCS) and press down
IFN processed mediates JAK-STAT signal paths and plays antiviral immune response effect.Researches show that nonsteroidal anti-inflammatory drug second
Acyl salicylic acid (ASA) targets NF-kB and lowers apoptosis developed by molecule, and the core of virus nucleoprotein is inhibited to export.Lung is atomized ASA, hair
Existing virus titer is decreased obviously, and extends the life span of mouse.Using special NF-kB inhibitor Bay11-7082 or
PDTC can inhibit the synthesis of viral gene vRNA, but on the output of the Nuclear Protein NP of virus without influence, this illustrates host factor
NF-kB is that influenza synthesizes needed for its vRNA.In the immune response that host copes with influenza infection, NF-kB is to participate in inflammation base
Because of the important adjusting transcription factor of expression.NF-kB not only transcribes pro-inflammation genes such as：TNF-a, IL-1 β, IL-6, IL-8, while
Adjust the expression of adhesion molecule (such as：ICAM-1, V-CAM-1, E-selectin), this shows NF-kB after immunocyte adherence
Migration aggregation to inflammation part plays an important role.But some researches show that NF-kB mediates the IL-1b generated and TNF-a difference
It can inhibit the activity of ENaC (sodium-ion channel) and destroy the close connection between alveolar epithelium, lead to the water retention in alveolar
Lead to the generation of acute respiratory distress syndrome ARDS with the damage at the interval of alveolar.However, also some researches show that influenza infections
The macrophage to lung is raised afterwards, has been activated NF-kB and PKR and has been mediated generation IFN-β jointly and raise and secrete antiapoptotic factors
TRAIL so that the damage of alveolar epithelium.
Influenza virus is by activating the transcription factor NF-kB of host so that itself is effectively replicated；It is activated in host cell
NF-kB mediates antiviral immunity reaction, but studies have reported that its immune response mediated leads to immunologic mjury.Therefore, with NF-
KB is that target spot not only reaches control influenza copy propagation, but also energy modulate host reply influenza immune response generates harmful damage, is
One strategy for preventing very well and treating influenza infection.
Invention content
The present invention provides application of the Aurantiamide acetate in the drug for preparing resisiting influenza virus, to for now clinically
The prevention and treatment of influenza provide a kind of effective drug.
Further, Aurantiamide acetate is answered in the drug for preparing the inflammatory reaction that influenza virus can be inhibited to induce
With.
Aurantiamide acetate has the following structure formula：
Further, Aurantiamide acetate is in the drug for preparing the inflammatory reaction that influenza A virus can be inhibited to induce
Using the cytopathic effect to influenza A virus mediation is in inhibiting effect.
Further, the Aurantiamide acetate can be obtained by commercial channel, can also be detached and be obtained from natural products
?.As a kind of specific implementation mode, Aurantiamide acetate can be detached from Radix Isatidis and be obtained, it is preferred that the Radix Isatidis is
Rhizoma et Radix Baphicacanthis Cusiae.
The present invention also provides a kind of drug for preventing or treating influenza infection, active constituent includes golden acyl
Amine alcohol ester, Aurantiamide acetate can be used as sole active agent, can also be combined with the substance of other medicinal licenses.
As a kind of specific implementation mode, pharmaceutically acceptable any dosage form can be made in the dosage form of the drug, be
The auxiliary material or carrier pharmaceutically allowed can be added in dosage form needed for preparing, the drug.Dosage form is, for example, tablet, hard capsule, soft
The dosage forms such as capsule, injection, granule, dripping pill.Required pharmaceutically acceptable carrier or auxiliary material is specific as pharmaceutically commonly used
Diluent and absorbent, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, carbon
Sour calcium, white bole, microcrystalline cellulose, alumina silicate etc.；Pharmaceutically common wetting agent and adhesive, such as water, glycerine, poly- second two
Alcohol, ethyl alcohol, propyl alcohol, starch slurry, dextrin, syrup, honey, glucose solution, mucialga of arabic gummy, gelatine size, carboxymethyl cellulose
Sodium, lac, methylcellulose, potassium phosphate, polyvinylpyrrolidone etc.；Pharmaceutically common disintegrant, such as dry starch, sea
Alginates, agar powder, laminaran, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, 12
Sodium alkyl sulfonate, methylcellulose, ethyl cellulose etc.；Disintegration inhibitor, for example, sucrose, glyceryl tristearate, cocoa butter,
Hydrogenated oil and fat etc.；Lubricant, such as talcum powder, silica, cornstarch, stearate, boric acid, atoleine, polyethylene glycol
Deng.It no longer enumerates about pharmaceutically acceptable carrier or auxiliary material, those of ordinary skill in the art can be according to being grasped
Common knowledge is specifically chosen.
As a kind of specific implementation mode, the Aurantiamide acetate in drug can be obtained by commercial channel, can also be led to
It crosses to detach from natural products and obtain.As a kind of specific implementation mode, Aurantiamide acetate can be detached from Radix Isatidis and be obtained,
Preferably, the Radix Isatidis is rhizoma et Radix Baphicacanthis Cusiae.
The method that the present invention also provides a kind of to detach Aurantiamide acetate from Radix Isatidis, this method comprises the following steps：
S1：It is by the ethanol water heating and refluxing extraction of chromatogram of Radix Isatidis volumetric concentration 60-90%, extracting solution is dense
Shorten medicinal extract into；Extraction time can be 1 time or repeatedly.
S2：By medicinal extract water dissolution at mixed liquor, water layer and chloroform layer are obtained with chloroform extraction mixed liquor, chloroform layer is depressurized
It is concentrated to give chloroform layer medicinal extract；
S3：Chloroform layer medicinal extract is added in silica gel, gradient elution is carried out with ethyl acetate-light petrol, collects containing gold
Removal solvent is concentrated under reduced pressure in the fraction of amide alcohol ester；
S4：By the product water dissolution of gained in step S3, it is added in reverse phase C18 columns, is first washed with water-methanol gradient
De-, then again with methanol-acetone gradient elution, collects the fraction containing Aurantiamide acetate, and removal solvent is concentrated under reduced pressure.
Preferably, further include step S5：The product obtained with water dissolution step S4, use volume ratio for 70: 30 acetonitrile-
Water carries out liquid phase preparation as eluant, eluent, the Aurantiamide acetate of purity >=90% is made, as a kind of specific implementation mode, liquid
It is Agilent 1100HPLC mutually to prepare liquid chromatograph used.
As a kind of specific implementation mode, when step S3, S4 is eluted, qualitative point is carried out to eluent with TLC
Analysis, required fraction is collected according to TLC analysis results.
As a kind of specific implementation mode, the Radix Isatidis is rhizoma et Radix Baphicacanthis Cusiae；
Preferably, the silica gel is purification on normal-phase silica gel.
Preferably, in step S3, ethyl acetate-light petrol carries out gradient elution according to volume ratio 9: 1-5: 1；As one kind
More preferably scheme, in step S3, ethyl acetate-light petrol is eluted according to five gradients, the volume ratio of five gradients
It is respectively successively：9:1,8:2,7:3,6:4,5:5.
Preferably, in step S4, gradient elution first is carried out according to the ratio of volume ratio 100: 0~0: 100 with water-methanol,
Then again with methanol-acetone carries out gradient elution according to the ratio of volume ratio 100: 0~50: 50.As still more preferably
Scheme in step S4, is eluted when being eluted with water-methanol according to 11 gradients, the volume ratio of water and methanol is respectively successively
100:0,90:10,80:20,70:30,60:40,50:50,40:60,30:70,20:80,10:90,0:100；Use methanol-acetone
It is eluted according to 6 gradients when elution, the volume ratio of methanol and acetone is 100: 0,90: 10,80: 20,70 respectively successively:
30、60∶40、50∶50。
Present inventor has found that rhizoma et Radix Baphicacanthis Cusiae compound E17 (Aurantiamide acetate) has resisiting influenza virus effect, right
The cytopathic effect that influenza A virus mediates is in inhibiting effect；
Present inventor has found that rhizoma et Radix Baphicacanthis Cusiae compound E17 (Aurantiamide acetate) has and inhibits host cell signal logical
The active effect in road；It is confirmed using stable transfection fluorescence report plasmid cell, rhizoma et Radix Baphicacanthis Cusiae compound E17 (Aurantiamide acetate)
It can inhibit the activation of NF-kB.It is confirmed using Western blot, rhizoma et Radix Baphicacanthis Cusiae compound E17 (Aurantiamide acetate) inhibits and stream
Feel the activation of the host inflammation associated signal paths NF-kB of induction.
It is confirmed using fluorescence real-time quantitative PCR, rhizoma et Radix Baphicacanthis Cusiae compound E17 (Aurantiamide acetate) can obviously inhibit
The H1N1 viruses infection A549 cellular inflammations factor (IL-6, IP-10, MCP-1, MIP-1 β, MIP-1a, IL-8, TNF-a, CCL-5,
IFN-b, IFN-lambda1, MIG) unconventionality expression and be in dose-dependence；And it further uses liquid-phase chip technology to exist
Protein level proves, rhizoma et Radix Baphicacanthis Cusiae compound E17 inhibit influenza infection A549 inductions inflammatory factor (IL-6, TNF-a, IP-10,
MCP-1, IL-8, Rantes) unconventionality expression.
Compared with existing drug, rhizoma et Radix Baphicacanthis Cusiae compound E17 (Aurantiamide acetate) is used to prepare anti influenza newtype drug
It has the advantages that：1, rhizoma et Radix Baphicacanthis Cusiae compound E17 (Aurantiamide acetate) action target is the signal point of host cell
It is sub rather than viral, antivirus action can be played, the effect for adjusting influenza induction excessive inflammation can be also played；2, compound
E17 (Aurantiamide acetate) targets the signaling molecule of host cell, is not likely to produce drug resistance；3, in a kind of preferred embodiment, gold
Color amide alcohol ester is that separation obtains monomer component from plant Baphicanthus cusia, and structure is clear, and chemical property is stablized, and quality control is easy to
System, toxic side effect are small.
Description of the drawings
Fig. 1 is the suppression to NF- к B signal pathway activateds using luciferase reporter gene technology detection rhizoma et Radix Baphicacanthis Cusiae compound E17
It makes and uses；
Fig. 2 is the effect that rhizoma et Radix Baphicacanthis Cusiae Compound Compound E17 inhibits influenza inducement signal signal pathway activated；
Fig. 3 is to detect the infection of rhizoma et Radix Baphicacanthis Cusiae compound E17 infected by influenza using real-time quantitative PCR (qRT-PCR) technology
The inflammatory factor gene expression of inducing host cell is horizontal；
Fig. 4 is to detect rhizoma et Radix Baphicacanthis Cusiae compound E17 infected by influenza senses in protein level using suspension liquid-phase chip technology
Contaminate the effect of the inflammatory response of inducing host cell；
Fig. 5 is using real-time quantitative PCR (qRT-PCR) technology, detection rhizoma et Radix Baphicacanthis Cusiae compound E17 infected by influenza infection
Afterwards inducing cell apoptosis and lead to the gene expression dose of the antiapoptotic factors TRAIL of body injury；
Fig. 6 is using Flow Cytometry, and induction A549 is thin after detection rhizoma et Radix Baphicacanthis Cusiae compound E17 infected by influenza infection
The effect of born of the same parents' apoptosis.
Specific implementation mode
In order to better understand the content of the present invention, present disclosure is made furtherly with reference to specific embodiment
It is bright, but the protection content of the present invention is not limited to following embodiment.
Aurantiamide acetate in following embodiment can be obtained by commercial channel, can also pass through following examples one
Method be made.
Embodiment one prepares rhizoma et Radix Baphicacanthis Cusiae E17 compounds (Aurantiamide acetate)
Step S1：Extraction
3Kg rhizoma et Radix Baphicacanthis Cusiae medicinal materials are cut into pieces, and fragment then uses the ethanol water of volumetric concentration 80% to heat back
Flow point be you can well imagine and be taken three times, and the volume of each ethyl alcohol is 30L, 24L, 18L, obtains ethanol extract, merge ethanol extract, heating
Ethyl alcohol is recovered under reduced pressure and obtains total extract medicinal extract.
Step S2：Solvent processing
Gained medicinal extract is mixed with water, and heating stirring, using the chloroform extraction gained mixed liquor of 1L obtain for 3 times water layer and
Chloroform layer then extracts gained water layer 3 times using 1L ethyl acetate, obtains ethyl acetate layer and water layer.Respectively merge chloroform layer and
Ethyl acetate layer is used in combination heating that solvent is recovered under reduced pressure and obtains chloroform layer medicinal extract and ethyl acetate layer medicinal extract.
Step S3：Purification on normal-phase silica gel processing
The chloroform layer medicinal extract of gained is added in silica gel, ethyl acetate-light petrol (volume ratio 9: 1-5: 1) gradient is used
It elutes, there are five gradients altogether for ethyl acetate-light petrol in the present embodiment, respectively are 9: 1,8: 2,7: 3,6: 4,5: 5, each
Gradient elution amount is 2L.It has been divided into 13 fractions by TLC display determinations, be used in combination to heat and solvent is recovered under reduced pressure to obtain 13 solids residual
Stay object.
Step 4：Reverse phase C18 processing
It is added in reverse phase C18 columns after No. 7 fraction (2.0g) water dissolutions, first uses water and methanol according to volume ratio 100: 0-0:
100 ratio carries out gradient elution, and in the present embodiment, water and methanol are specifically to carry out gradient according to following volume ratio successively to wash
It is de-：100: 0,90: 10,80: 20,70: 30,60: 40,50: 50,40: 60,30: 70,20: 80,10: 90,0: 100, each gradient
Elution amount is 1L；Then again with methanol and acetone carry out gradient elution, this implementation according to the ratio of volume ratio 100: 0-50: 50
In example, methanol and acetone are specifically to carry out gradient elution according to following volume ratio successively：100:0,90:10,80:20,70:30,
60: 40,50: 50, each gradient elution amount is 1L；In elution process, is determined by TLC displays and be divided into 12 fractions.
Step S5：Half preparation is handled
7-8 number fractions (63.0mg) collected in step S4 are with after water dissolution, using acetonitrile-water (volume ratio 70: 30)
Liquid phase preparation is carried out on Agilent 1100HPLC for eluant, eluent, and compound E17 (4.1mg), purity >=90% is made.
Products therefrom is detected, under data enter：
Compound (E17)1H-NMR (600MHz, CDCl3)δ：4.35 (1H, m, H-2), 2.80 (2H, heptet, H-3),
7.20 (2H, m, H-5, H-9), 7.07 (2H, d, J=8.1Hz, H-6, H-8), 7.16 (1H, m, H-7), 3.93 (1H, dd, J=
11.3,4.9Hz, H-10b), 3.82 (1H, dd, J=11.3,4.2Hz, H-10a), 2.03 (3H, s, H-12), 4.76 (1H, q, J
=5.82Hz, H-13), 7.72 (2H, d, J=7.77Hz, H-16, H-20), 7.44 (2H, t, J=7.81Hz, H-17, H-19),
7.53 (1H, t, J=7.46Hz, H-18), 3.21 (1H, dd, J=13.7,5.91Hz, H-21b), 3.06 (1H, dd, J=
13.7,8.5Hz, H-21a), 7.31 (2H, m, H-23, H-27), 7.28 (2H, m, H-24, H-26), 7.25 (1H, m, H-25)
.13C-NMR (150MHz, CDCl3)δ：170.3 (C-1), 49.5 (C-2), 37.4 (C-3), 136.6 (C4), 128.7 (C-5),
128.8 (C-6), 126.8 (C-7), 128.8 (C-8), 128.7 (C-9), 64.6 (C-10), 170.8 (C-11), 20.4 (C-
12), 55.0 (C-13), 167.1 (C-14), 133.6 (C-15), 127.0 (C-16), 128.6 (C-17), 131.9 (C-18),
128.6 (C-19), 127.0 (C-20), 38.4 (C-21), 136.7 (C-22), 128.1 (C-23), 128.3 (C-24), 127.2
(C-25), 128.3 (C-26), 128.1 (C-27).
E17 structure elucidations：
E17 is white crystal.1H-NMR and1H-1It is ABX systems that H-COSY, which prompts the compound,：δ 4.76 (1H, q, J=
5.8Hz, H-13), δ 3.21 (1H, dd, J=13.7,5.9Hz, H-21b), δ 3.06 (1H, dd, J=13.7,8.5Hz, H-
21a)；δ 4.35 (1H, m, H-2), δ 3.93 (1H, dd, J=11.3,4.9Hz, H-10b) δ 3.82 (1H, dd, J=11.3,
4.2Hz, H-10a).It is δ 4.76 (1H, q, J=5.8Hz, H-13) and nitrogen hydrogen group chemistry to also show hydrogen chemical shifts simultaneously
Displacement is δ 6.76 (1H, d, J=7.7Hz, N-Hb) and hydrogen chemical shifts are δ 4.35 (1H, m, H-2) and another two group (δ
2.80 (2H, m) and δ 5.95 (1H, d, J=8.4Hz, N-Ha)) contact.The compound, which contains, to be obtained to the analysis of aromatic series hydrogen
Three AA ' BB ' C, are each single substituted benzene rings.13C-NMR and13C-DEPT135 is then prompted there are two the compound contains
Amino (δ C170.3 and 167.1), an ester group (8C 170.8), two benzylidenes (δ C 37.4 and 38.4), an aldehyde radical
(δ C64.6), a methyl (δc20.4), 17 methylene and trimethyl.All above evidences show that the compound is gold
Color amide alcohol ester.
Its structural formula is as follows：
Two cytopathic effect method (CPE) of embodiment detects the antivirus action of rhizoma et Radix Baphicacanthis Cusiae compound E17
1. experiment material：
1.1 drug given the test agent
Rhizoma et Radix Baphicacanthis Cusiae compound E17 (preparation of embodiment one), it is 90% to detect its purity through HPLC.Positive control medicine：
Oseltamivir Oseltamivir.
1.2 cell
Dog kidney cells (MDCK) is quoted from the American Type Culture Collection committee of Chinese Academy of Sciences cell bank.
1.3 Strain
H1N1virus PR8 plants (A/PR/8/34, H1N1), ichi plants of (A/HK/8/ of A type H3N2 influenza virus As
68, H3N2), A type 09H1N1 influenza viruses (A/GZ/GIRD07/09 (H1N1)), influenza B virus (B/Lee/1940
(FluB)) it is this room clinical separation strain, bird flu H9N2 (A/HK/Y280/97, H9N2) is old by College of Veterinary Medicine, South China Agricultural University
New professor is built to give.
2. experimental method
Include the following steps：
1) MDCK cell monolayers are inoculated with 96 orifice plates and are washed 1 time with PBS；
2) 100 TCID50 difference strains are added later：(A/PR/8/34, H1N1), (A/HK/8/68, H3N2), (A/
GZ/GIRD07/09 (H1N1)), (B/Lee/1940 (FluB), (A/HK/Y280/97, H9N2) virus liquid adsorb MDCK respectively
Cell, 100 holes μ L/, 37 DEG C are incubated 2 hours；
3) virus incubation liquid is discarded, the holes DMEM culture solution 100uL/ of the 1.5ug/mLTPCK containing final concentration, an A rows left side 6 is added
A multiple holes are not added with Virus culture base, but the culture medium of 200uL drug containing E17 and positive control medicine, drug E17 highests is added
A concentration of 500ug/ml from top to bottom uses volley of rifle fire multiple proportions gradient dilution, result is observed after 48 hours later.
4) 6 grades of standards of cytopathy (CPE) and the antiviral activity testing number of rhizoma et Radix Baphicacanthis Cusiae compound E17 caused by virus
According to respectively as shown in the following table 1,2：
6 grades of standards of cytopathy (CPE) caused by 1 virus of table
| Cellular morphology | Extent of disease |
| Cell growth is normal, and no lesion occurs | - |
| Cytopathy is less than the 10% of entire cell monolayer | ± |
| Cytopathy accounts for about the 25% of entire cell monolayer | + |
| Cytopathy accounts for about the 50% of entire cell monolayer | 2+ |
| Cytopathy accounts for about the 75% of entire cell monolayer | 3+ |
| Cytopathy accounts for about 75% or more of entire cell monolayer | 4+ |
5) after observing and recording CPE results, rhizoma et Radix Baphicacanthis Cusiae compound E17 and positive control are calculated according to Reed-Muench methods
TC50, IC50 and SI (SI=TC50/IC50) of the object Oseltamivir to different influenza strains.
The antiviral activity of 2 rhizoma et Radix Baphicacanthis Cusiae compound E17 of table
SI > 2：It is less toxic efficient；1 < SI < 2：High poison is inefficient；SI < 1：In vain
3. experimental result
Cytopathic effect method (CPE) testing result is as shown in table 2, and E17 is to influenza A virus for rhizoma et Radix Baphicacanthis Cusiae compound
Different virus strain：A/PR/8/34 (H1N1), A/GZ/GIRD07/09 (H1N1), A/HK/8/68 (H3N2) have antiviral work
With.
Three luciferase reporter gene technology of embodiment detects suppressions of the rhizoma et Radix Baphicacanthis Cusiae compound E17 to NF-kB signal pathway activateds
It makes and uses
1. experiment material：
1.1 drug given the test agent
Rhizoma et Radix Baphicacanthis Cusiae compound E17 (preparation of embodiment one).
1.2 cell
The HEK293T cells for having stablized corotation NF- κ B-TATA-F-LUCI and pQCXIP-eGFP plasmids breathe disease by Guangzhou
Sick National Key Laboratory doctor Li Chufang give.(document：Li J, Zhou B, Li C, Chen Q, WangY, Li z, Chen
T, Yang C, Jiang Z.Zhong N et al：Larieiresinol-4-O-beta-D-glucopyranoside from
the root of Isatis indigotica inhibits influenza A virus-induced pro-
Inflammatory response.JEthnopharmacol 2015,174：379-386.)
1.3 reagent
Luciferase Assay System (article No.：E2610 is purchased from promega companies), recombined human cytokine TNF-a is purchased from
Promega companies
2. experimental method, such as following：
1) HEK293T cells have stablized corotation NF- κ B-TATA-F-LUCI and pQCXIP-eGFP plasmids, the cell line by
NF-kB start expression reporter gene luciferase and sustainable expression Egfp as internal reference, (cell line is by Guangzhou respiratory disorder
National Key Laboratory doctor Li Chufang give).
2) HEK293T cells are with 5X104Concentration is inoculated into 96 orifice plates, in 37 DEG C of temperature, 5%CO2Incubator in train overnight
It supports；
3) after cell is adherent, setting experimental group is respectively：Control group, TNF-a groups (20ng/ml) are (public purchased from peprotect
Department), TNF-a+E17 (30ug/ml) group, TNF-a+E17 (60ug/ml), TNF-a+E17 (90ug/ml) group, independent E17 groups
(60ug/ml) group.Discard cell culture supernatant, be added in setting experimental group corresponding reagent or (and) drug；
4) it after for 24 hours, discards culture supernatant and cell is resuspended using PBS, cell will be resuspended and be divided into two parts, a copy of it is added steady
Determine the expression of Luciferase Assay System (E2610, promega company) detection luciferase, while another detection GFP fluorescence
Intensity, the ratio to detect luciferase luminous intensity and internal reference EGFP carry out evaluation drug E17 and make to the inhibition of NF-KB accesses
With.
3. experimental result
As a result referring to Fig. 1, luciferase reporter gene detection finds that rhizoma et Radix Baphicacanthis Cusiae compound E17 is to NF- κ B signal pathway activateds
It is inhibited, and be in dose-dependence.
Example IV rhizoma et Radix Baphicacanthis Cusiae compound E17 inhibits influenza inducement signal signal pathway activated
1. experiment material：
1.1 drug given the test agent
Rhizoma et Radix Baphicacanthis Cusiae compound E17 (preparation of embodiment one).
1.2 cell
A549 cells are purchased from ATCC, are stored in this laboratory；
1.3 chemical reagent and antibody
DMEM/DF12 (1: 1) culture medium is purchased from Gibco companies；Tris base are purchased from Ameresco companies；Sodium chloride
(NaCl) it is purchased from Guangzhou Chemical Reagent Factory；Potassium chloride (KCl) is purchased from Guangzhou Chemical Reagent Factory；Potassium dihydrogen phosphate (KH2PO4) be purchased from
Guangzhou Chemical Reagent Factory；Disodium hydrogen phosphate (Na2HPO4·12H2O) it is purchased from Guangzhou Chemical Reagent Factory；Glycine Glycine is purchased from
Guangzhou Chemical Reagent Factory；Dodecyl sodium sulfate (SDS) is purchased from sigma companies of the U.S.；Tween Tween-20 is purchased from Guangzhou chemistry
Chemical reagent work；BCA determination of protein concentration kits are purchased from Thermo companies of the U.S.；RIPA lysates are 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；Ammonium Persulfate 98.5 (APS) is purchased from Ameresco companies of the U.S.；
Pvdf membrane is purchased from Life science companies of the U.S.；Cocktail (protease inhibitors) is purchased from sigma companies of the U.S.；Defatted milk
Powder is purchased from Gibco companies；Fetal calf serum is purchased from Gibco companies；TEMED (N, N- methylene-bisacrylamide) is purchased from U.S. Bio-
Rad companies；Rabbit-anti people's p-p65 antibody is purchased from CST companies of the U.S.；Rabbit-anti people's p65 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's ERK antibody is purchased from CST companies of the U.S.；Rabbit-anti people's p-p38 antibody is purchased from the U.S.
CST companies；Rabbit-anti people's p38 antibody is purchased from CST companies of the U.S.；Rabbit-anti people's 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..
2. experimental method, such as following：
1) the A549 cells in T75 bottles are digested with pancreatin, after cell all falls off, is added and contains 10% in right amount
DMEM/DF12 (1: 1) culture medium of FBS (volume ratio) terminate the effect of pancreatin；
2) cell digested is transferred in the centrifuge tube of 15ml, carries out centrifugation 1000rpm × 5min；
3) DMEM/DF12 containing 10%FBS (1: 1) culture medium in right amount is added cell is resuspended, the density for adjusting cell will
Cell is laid in 6 orifice plates, is positioned in incubator, for use after culture 12h cells are adherent；
4) setting of experimental group is carried out, including：Normal group；Influenza virus group, low-dose drugs intervention group (influenza virus+
E17 (30 μ g/ml)), middle dosage pharmaceutical intervention group (influenza virus+E17 (60 μ g/ml)), high dose medicament intervention group (influenza disease
Poison+E17 (90 μ g/ml)), drug alone group (E17 (60 μ g/ml)).A549 cells are taken out, original culture medium is discarded, adds
Enter PBS to wash twice, then be added (H1N1) containing influenza virus A/PR8/3/4 (MOI=0.1) serum-free DMEM/DF12 (1:
1) culture medium adherent cell 2h；
5) cell is washed with PBS and removes unadsorbed virus, E17 (the 30 μ g/ of various concentration are added in medicine group intervention group
Ml, 60 μ g/ml, 90 μ g/ml), the E17 of 60 μ g/ml is added in drug alone group.After continuing culture 24 hours；Cell 6 orifice plates are set
In on ice, cells and supernatant is discarded, cold PBS is used in combination to wash twice；Washing finishes, and is rapidly added 130ul cell pyrolysis liquids
RIPA (containing protease inhibitors cocktail, is added with cell pyrolysis liquid RIPA volume ratios (V/V) for 1: 100 and dense eventually
Degree is the PMSF of 10uM), extract total protein of cell.
6) 1.5mlEP pipes are marked and they is placed on ice, cell lysate is transferred to the EP for having marked each group
Pipe, and 30min is shaken on shaking table, allow albumen fully to crack；
7) centrifuge that cell lysate is moved to 4 DEG C of precoolings carries out 13000rpm × 15min centrifugations；
8) centrifugation is finished, and the precipitation of lysate is discarded, and supernatant is carried out packing and is stored in -80 DEG C；
The albumen concentration of cell total extract is measured, configuration mark is carried out by BCA protein detection kits specification
Quasi- product take 96 orifice plates, and the standard items and sample (2 multiple holes) in the holes 10ul/ are added, and BCA is then hole-specifically added and detects working solution
The holes 200ul/ (configuration work liquid A: B=1: 50), 37 DEG C of incubation 30min after mixing, take out 96 orifice plates and are cooled to room temperature, in enzyme mark
On instrument OD values are measured in absorbance 572nm.Standard curve is drawn, sample OD values are converted into concentration.
9) preparation of sds page：
10%SDS polyacrylamide gel electrophoresis separation gels are first configured according to formula：
| Composition | Volume |
| ddH2O | 4ml |
| 30%ACr (acrylamide) | 3.3ml |
| 1.5M Tris.HCl (PH=8.8) | 2.5ml |
| 10%SDS | 0.1ml |
| 10%APS (ammonium persulfate) | 0.1ml |
| TEMED | 0.004ml |
Separation gel is perfused after mixing well between separation of glasses plate, perfusion is finished is added ddH in top layer2O seals separation
Glue liquid surface waits for 30min, separation gel condensation intact；
10) according to formula, first configuration 5%SDS polyacrylamide gel electrophoresises concentrate glue：
| Composition | Volume |
| ddH2O | 3.4ml |
| 30%ACr (acrylamide) | 0.83ml |
| 1M Tris.HCl (PH=6.8) | 0.63ml |
| 10%SDS | 0.05ml |
| 10%APS (ammonium persulfate) | 0.05ml |
| TEMED | 0.005ml |
Insertion comb is finished in perfusion concentration glue, perfusion between separation of glasses plate after mixing well, and 30min, concentration is waited for be gelled
It ties intact, can use.
10) albuminate：With 5xSDS polyacrylate hydrogels sample loading buffer (5x loading buffer) and sample (20 μ
G) in the ratio (1: 4) of volume and mass ratio (u1/ug)) mixing, 95 DEG C are denaturalized 10min, and it is anti-to be immediately passed to cooled on ice 5min
Only protein renaturation.
11) it is loaded：3000rpm × 1min is centrifuged, is hole-specifically loaded；
12) electrophoresis：With Bio-Rad vertical electrophoresis apparatus, 90V constant pressures electrophoresis about (1-1.5) h waits for that bromophenol blue indicator reaches
Stop electrophoresis when separation gel bottom；
12) Western transferring films：Advance methanol impregnates 15min and activates pvdf membrane.Gel is removed after electrophoresis, excision is more
Remaining Blank gel cuts 6 Whatman3M filter paper after trimming by its size.By 3 filter paper-gel-filter paper of pvdf membrane -3
Sequence stack neatly be placed on it is wet turn on negative plate, with the bubble between clean glass bar venting gel and pvdf membrane, in freezer
In, with the electric transferring film 2h of the perseverance of 380mA.
13) it closes：After transferring film, takes out film and 5% skimmed milk power (5%milk/TBST) is added and close pvdf membrane simultaneously
In jog 2h on room temperature shaker；
13) antibody dilution and incubation：Configure 5%BSA/TBST, by purchased from CST companies antibody NF-kB, P38MAPK,
The corresponding ratio of ERK1/2MAPK, GAPDH specification dilutes NF-kB, P38MAPK respectively, and ERK1/2MAPK, GAPDH antibody add
Enter on the pvdf membrane finished to transferring film, is placed in Cool Room 4 DEG C and is incubated overnight；
14) it second day, takes out pvdf membrane and restores to room temperature, remove primary antibody and wash film 3 times, 5min/ times with washing lotion TBST.
The goat-anti rabbit secondary antibody being coupled with horseradish peroxidase (HRP) that (V/V) 1: 1000 is diluted by volume, room is then added
Temperature is incubated 1h.It removes secondary antibody and washes film 3 times, 5min/ times with washing lotion TBST；
15) film is taken out, drip-dry moisture on filter paper is added ECL immunofluorescences and shines color developing agent；
16) tabletting is carried out in darkroom, and fixes film in developing liquid developing and fixing solution；
17) interpretation of result and arrangement.
3. experimental result
As a result see Fig. 2.Immunoblot results are shown, after influenza A/PR8/3/4 (H1N1) infects A54924h, cell signal
Access NF-kB, P38MAPK, ERK1/2MAPK are obviously activated.Rhizoma et Radix Baphicacanthis Cusiae compound E17 obviously inhibits influenza A/PR8/3/4
(H1N1) activation of people's A549 cell-signaling pathways NF- κ B signal accesses is induced.
The influence of the five infection induced host inflammation gene expression of rhizoma et Radix Baphicacanthis Cusiae compound E17 infected by influenza of embodiment
1. experiment material：
1.1 drug given the test agent
Rhizoma et Radix Baphicacanthis Cusiae compound E17 (preparation of embodiment one).
1.2 cells and strain
A549 cells are purchased from ATCC, are stored in this laboratory；PR8 plants of H1N1virus (A/PR/8/34,
H1N1), it is stored in this laboratory.
1.3 reagent
Reverse transcription reaction kit (is purchased from Takara companies；Article No.：RR036A)；(the purchase of real-time quantitative PCR reaction kit
From Takara companies；Article No.：RR390A)
2. experimental method, such as following：
1) by A549 cells with 5X106Cell density kind to 6 orifice plates, be positioned over 37 DEG C of temperature, 5%CO2Incubator
In；
2) be incubated overnight cell it is adherent after, discard former culture medium, PBS be added and washs 2 times.Then be added containing influenza virus A/
DMEM/DF12 (1: 1) culture medium adherent cell 2h of PR8/3/4 (H1N1) serum-free；Then, cell is washed into removing with PBS
Unadsorbed virus；
3) experimental group is set as viral group, pharmaceutical intervention group, and wherein pharmaceutical intervention group uses mode of drug, medicine group
Various concentration E17 (30 μ g/ml, 60 μ g/ml, 90 μ g/ml) is added in intervention group (corresponding to H1N1+E17 in Fig. 3)；Viral group of (figure
Correspond to H1N1 in 3) it is not added with drug and is intervened.It is additionally provided with drug alone group (corresponding to E17 in Fig. 3), A549 cells are not
Through viruses adsorption, the E17 of 60 μ g/ml is added.
4) it after continuing culture 24 hours, discards in 6 orifice plates after cells and supernatant, cold PBS is washed 2 times, and 1ml is added
Trizol (Life technologies companies) is simultaneously incubated at room temperature 5min；It is (optional：Or go in 1.5mlEP pipes, in-
It is preserved in 80 ° of refrigerators；Or directly extract)；
5) it subsequently carries out in accordance with the following steps：
1. 200ul chloroforms are added, 15sec is shaken, 2-3min is incubated in room temperature；It is placed in 4 ° of centrifuges and is centrifuged：
13000rpm x 15min；
2. centrifugation is finished, supernatant, which is transferred to new 1.5EP, manages, and 500ul isopropanols are added；It is incubated 10min at room temperature；It is placed in
It is centrifuged in 4 DEG C of centrifuges：12000rpm x 10min；
3. centrifugation is finished, 75% ethyl alcohol of 1ml is added and washed once, is placed in 4% centrifuge and is centrifuged：7500rpm x
10min；
4. discarding supernatant, it is placed at room temperature for 5-10min, is added without RNase water dissolution RNA, carries out reverse transcription at once into cDNA.
5. measuring the concentration (ng/ μ l) of total serum IgE in spectrophotometer and calculating the volume of total serum IgE：Quasi- addition reverse transcription is anti-
It is 1000ng to answer the amount of total serum IgE in system, then calculates the volume V=1000/RNA concentration of required total serum IgE；
MRNA reverse transcriptions are at cDNA, the following (kit of configuration reverse transcription reaction system：Takara RR036A)：
6. carrying out reverse transcription reaction, reaction condition is as follows：
37℃ 15min
85℃ 5Sec
4℃ ∞
Sample is collected to carry out subsequent quantitation PCR experiment or be stored in -20 DEG C.
8. real-time quantitative PCR reaction kit (article No.：Takara RR390A)
It is as follows to configure reaction system：
Carry out Real time PCR reaction conditions (ABI7500)
Primer sequence and probe sequence are as follows：
3. experimental result
As a result such as Fig. 3 is shown, after influenza A/PR8/3/4 (H1N1) infects the progress drug E17 interventions of A549 cells, hence it is evident that suppression
Inflammatory factor IL-6, IL-8, IP-10, MCP-1, MIP-1 α, the MIP-1 β of influenza processed induction, MIG, IFN-β, the base of IFN- λ 1
Because of horizontal expression.
Six Bio-plex Suspension array techniques of embodiment are lured in protein level detection rhizoma et Radix Baphicacanthis Cusiae E17 infected by influenza infection
Lead the effect of the inflammatory factor secretion of host cell
1. experiment material：
1.1 drug given the test agent
Rhizoma et Radix Baphicacanthis Cusiae compound E17 (preparation of embodiment one).
1.2 cells and strain
A549 cells are purchased from ATCC；H1N1virus PR8 plants (A/PR/8/34, H1N1), is stored in this experiment
Room.
1.3 reagent
Bio-Plex suspension chip inspecting reagent unit (article No.s：Y500CR7L16)
2. experimental method, such as following：
1) by A549 cells with 5X106Cell density kind to 6 orifice plates, be positioned in incubator, be incubated overnight；
2) after cell is adherent, former culture medium is discarded, PBS is added and washes twice, is then added and contains influenza virus A/PR8/3/4
(H1N1) DMEM/DF12 (1: 1) culture medium adherent cell 2h of serum-free；Then, with PBS by cell wash remove it is unadsorbed
Virus；
3) Control (control group) (normal A549 cells), viral group, pharmaceutical intervention group are set；Wherein, pharmaceutical intervention group is adopted
Rhizoma et Radix Baphicacanthis Cusiae compound E17 (30 μ g/ml, 30 μ g/ml, 90 μ of various concentration are added in drug treatment pattern, pharmaceutical intervention group
g/ml)；Viral group is not added with pharmaceutical intervention.It is additionally provided with drug alone group, 60 μ g/ are added without viruses adsorption in A549 cells
The rhizoma et Radix Baphicacanthis Cusiae compound E17 of ml；
4) after continuing culture 24 hours, cells and supernatant is moved into 1.5mlEP pipes.Then, it is placed in the centrifugation of 4 DEG C of precoolings
The centrifugation of 13000rpm x 15min is carried out in machine.Centrifugation is finished, and discards the cell fragment of precipitation, and supernatant is carried out aliquot point
Dress, then freezes in -80 ° of refrigerators, avoids multigelation, used when to be detected.
5) Inflammatory Factors Contents in Bio-plex Suspension array techniques detection cell conditioned medium：
1. cytokines measurement is tested：Bio-Plex suspensions chip inspecting reagent unit (Bio Rad companies) is taken out, keeps it flat
Weighing apparatus restores to room temperature.Meanwhile it taking out sample and slowly being thawed on ice.
2. the position of standard sample wells on flat underside (96 orifice plate), blank well, the holes control and sample well, standard items are arranged
2 multiple holes in hole and blank well.
3. (Bio-Plex suspensions chip inspecting reagent unit) ratio is added to detection buffer solution assay to specifications
Antibody microballoon is diluted in buffer, vortex homogeneous magnetic attracts microballoon, and flat underside is added to per hole 50ul microballoon mixed liquors
In.
4. 100ul dcq buffer liquid wash buffer are added per hole, and flat underside is placed on magnetic frame and carries out washing magnetic
Property attract microballoon, wash 2 times.
5. being loaded：The standard items being serially diluted, ready sample are added separately to standard sample wells, the sample of flat underside
In sample wells, assay buffer are added in blank well, with sealed membrane sealing plate.
6. being incubated：It is wrapped with aluminium foil, at room temperature, flat underside is incubated 30min in 96 plate shaker 300rpm.
7. washing：Incubation finishes, and flat underside is placed on magnetic frame and discards liquid.100ul wash are added per hole
Buffer, and flat underside is placed on magnetic frame and discards liquid, it repeats this process and washs 3 times.
8. plus detection antibody：Detection antibody is shaken up using preceding vortex, ratio is added to inspection to specifications by detection antibody
It surveys in antibody diluent, the uniform inflammatory factor IL-6, IL-8 that is vortexed, IL-10, IFN-γ, IP-10, MCP-1 (MCAF),
25ul is added per hole in flat underside for the detection antibody of RANTES, TNF-a.At room temperature, flat underside is placed in 96 plate shakers
300rpm is incubated 30min.
9. washing：Incubation finishes, and flat underside is placed on magnetic frame and discards liquid, and 100ul wash are added per hole
buffer.And flat underside is placed on magnetic frame and discards wash buffer, it repeats this process and washs 3 times.
10. the Streptavidin of 50ul fluoresceins PE labels is added per hole in flat underside, at room temperature, in 96 plate shakers
300rpm is incubated 10min.
Washing, incubation finish, and flat underside are placed on magnetic frame and discards liquid, and 100ul wash are added per hole
buffer.It is placed on magnetic frame and discards wash buffer, wash repeatedly 3 detections
125ul is added per hole in flat underside and detects buffer solution assay buffer, is protected from light rocker 1100rpm*30 at room temperature
Second, it is detected with Bio-plex 200.
3. experimental result
The results are shown in Figure 4, and rhizoma et Radix Baphicacanthis Cusiae compound E17 obviously inhibits the inflammatory factor that influenza virus induces after intervening
The level of (IL-6, TNF-alpha, IP-10, MCP-1, IL-8, Rantes).The E17 interventions of prompt rhizoma et Radix Baphicacanthis Cusiae compound can adjust
Throttling is felt infection induced excessive inflammation and is answered.
The expression shadow of the six infection induced host's apoptogene TRAIL of rhizoma et Radix Baphicacanthis Cusiae compound E17 infected by influenza of embodiment
It rings
1. experiment material：
1.1 drug given the test agent
Rhizoma et Radix Baphicacanthis Cusiae compound E17 (preparation of embodiment one).
1.2 cells and strain
A549 cells are purchased from ATCC；H1N1virus PR8 plants (A/PR/8/34, H1N1), is stored in this experiment
Room.
1.3 reagent
Reverse transcription reaction kit (is purchased from Takara companies；Article No.：RR036A)；(the purchase of real-time quantitative PCR reaction kit
From Takara companies；Article No.：RR390A)
2. experimental method, such as following：
1) by A549 cells with 5X106Cell density kind to 6 orifice plates, be positioned over 37 DEG C of temperature, 5%CO2Incubator
In；
2) be incubated overnight cell it is adherent after, discard former culture medium, PBS be added and washs 2 times.Then be added containing influenza virus A/
DMEM/DF12 (1: 1) culture medium adherent cell 2h of PR8/3/4 (H1N1) serum-free；Then, cell is washed into removing with PBS
Unadsorbed virus；
3) experimental group is set as viral group, pharmaceutical intervention group, and wherein pharmaceutical intervention group uses mode of drug, medicine group
Various concentration E17 (30 μ g/ml, 60 μ g/ml, 90 μ g/ml) is added in intervention group (PR8+E17 is corresponded in Fig. 5)；Viral group is not added with
Drug is intervened.It is additionally provided with drug alone group, the E17 of 60 μ g/ml is added without viruses adsorption in A549 cells.
4) it after continuing culture 24 hours, discards in 6 orifice plates after cells and supernatant, cold PBS is washed 2 times, and 1ml is added
Trizol (Life technologies companies) is simultaneously incubated at room temperature 5min；It is (optional：Or go in 1.5mlEP pipes, in-
It is preserved in 80 ° of refrigerators；Or directly extract)；It carries out in accordance with the following steps later：
Cell total rna extraction, mRNA reverse transcriptions are at cDNA and real-time quantitative PCR reaction with reference to the corresponding of embodiment five
Step carries out；Wherein, the present embodiment antiapoptotic factors TRAIL primer sequences and probe sequence used in real-time quantitative PCR reaction
Row are as follows：
3. experimental result
The results are shown in Figure 5, and the gene expression dose of antiapoptotic factors TRAIL is bright afterwards for 24 hours for influenza infection A549 cells
Aobvious up-regulation, rhizoma et Radix Baphicacanthis Cusiae compound E17 obviously inhibit the gene expression water of the antiapoptotic factors TRAIL of influenza virus induction after intervening
It is flat, and be in dose-dependence.Rhizoma et Radix Baphicacanthis Cusiae compound E17 is prompted to intervene the body damage caused by inhibiting influenza infection apoptosis-induced
Wound.
It is apoptosis-induced that eight Annexin V detection rhizoma et Radix Baphicacanthis Cusiaes E17 of embodiment intervenes influenza
1. experiment material：
1.1 drug given the test agent
Rhizoma et Radix Baphicacanthis Cusiae compound E17 (preparation of embodiment one).
1.2 cells and strain
A549 cells are purchased from ATCC, are stored in this laboratory；H1N1virus PR8 plants (A/PR/8/34, H1N1)
1.3 reagent
Annexin V apoptosis detection kit (is purchased from affymetrix ebioscience companies, article No.：88-8005)
2. experimental method, such as following：
1) the A549 cells in T75 bottles are digested with pancreatin, after cell all falls off, be added appropriate culture medium into
Row terminates the effect of pancreatin；
2) cell digested is transferred in the centrifuge tube of 15ml, carries out centrifugation 1000rpmx5min；
3) culture medium is added cell is resuspended, cell is laid in 6 orifice plates by the density for adjusting cell, placement and incubator
In, it is for use after culture 12h cells are adherent；
4) setting of experimental group is carried out, including：Normal group；Influenza virus group；Pharmaceutical intervention group：Influenza virus+E17
(60ug/ml) group, influenza virus group+E17 (90ug/ml) group；
5) A549 cells are taken out, discards original culture medium, PBS is added and washes twice, influenza virus group and drug are dry
Pre- group is added the culture medium adherent cell 2h containing influenza virus A/PR8/3/4 (H1N1) serum-free after PBS washings, and normal group is not
Do the processing；
6) cell is washed with PBS and removes unadsorbed virus, the drug of various concentration, influenza disease is added in pharmaceutical intervention group
Poison group and normal group are then substituted with PBS, continue culture 24 hours；
7) then, cell 6 orifice plates are placed on ice, discard cells and supernatant, cold PBS is used in combination to wash twice；
8) washing finishes, and the trypsin digestion cell without EDTA is added；And cell is moved to the centrifuge tube of 1.5ml
It is centrifuged in 4 DEG C of centrifuges, 1000rpm x 3min, centrifugation, which is finished, to be discarded supernatant；
9) it is primary that PBS washing cells are added, 4 DEG C of centrifugation 1000rpm x 3min；
10) 1xbinging buffer are added washed once, 4 DEG C of centrifugation 1000rpm x 3min；
11) cell is resuspended in the cell suspension that 500ul is added, and prepares cell suspension；
12) 5ul Annexin V and 5ul Propidium iodide staining cells are added, room temperature, which is protected from light, stands 15min；
13) 1xbinging buffer washed once；
14) machine is analyzed on
3. experimental result
The results are shown in Figure 6, and rhizoma et Radix Baphicacanthis Cusiae E17 inhibits withering for influenza A/PR8/3/4 (H1N1) infection induced A549 cells
It dies.
By above-mentioned experimental result it is found that rhizoma et Radix Baphicacanthis Cusiae compound E17 (Aurantiamide acetate) has resisiting influenza virus effect,
It is in inhibiting effect to the cytopathic effect that influenza A virus mediates；Rhizoma et Radix Baphicacanthis Cusiae compound E17 can inhibit the work of NF-kB
Change.It is confirmed using Western blot, rhizoma et Radix Baphicacanthis Cusiae compound E17 inhibits the host inflammation associated signal paths with influenza induction
The activation of NF-kB.It is confirmed using fluorescence real-time quantitative PCR, rhizoma et Radix Baphicacanthis Cusiae compound E17 can obviously inhibit H1N1 viruses to infect
The A549 cellular inflammations factor (IL-6, IP-10, MCP-1, MIP-1 β, MIP-1a, IL-8, TNF-a, CCL-5, IFN-b, IFN-
Lambdal, MIG) unconventionality expression and be in dose-dependence；And it further uses liquid-phase chip technology to demonstrate,prove in protein level
It is bright, rhizoma et Radix Baphicacanthis Cusiae compound E17 inhibition influenza infection A549 inductions inflammatory factor (IL-6, TNF-a, IP-10, MCP-1, IL-8,
Rantes unconventionality expression).
Compared with existing drug, rhizoma et Radix Baphicacanthis Cusiae compound E17 (Aurantiamide acetate) action target is the letter of host cell
Number molecule rather than it is viral, antivirus action can be played, can also play the effect for adjusting influenza induction excessive inflammation；Compound
E17 targets the signaling molecule of host cell, is not likely to produce drug resistance；In as a preferred embodiment, Aurantiamide acetate be from
Separation obtains monomer component in plant Baphicanthus cusia, and structure is clear, and chemical property is stablized, and is easy to quality control, toxic side effect is small.
It is not specified in text, it is common knowledge or routine techniques hand of the those of ordinary skill in the art according to its grasp
Section 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, in the range of still falling within technical solution of the present invention.
Claims (8)
1. a kind of method detaching Aurantiamide acetate from Radix Isatidis, which is characterized in that include the following steps：
S1：By the ethanol water heating and refluxing extraction of chromatogram of Radix Isatidis volumetric concentration 60-90%, extracting solution is condensed into leaching
Cream；
S2：By medicinal extract water dissolution at mixed liquor, water layer and chloroform layer are obtained with chloroform extraction mixed liquor, chloroform layer is concentrated under reduced pressure
Obtain chloroform layer medicinal extract；
S3：Chloroform layer medicinal extract is added in silica gel, with ethyl acetate-light petrol gradient elution, collection contains Aurantiamide acetate
Fraction, be concentrated under reduced pressure removal solvent；
S4：It is added to the product water dissolution of gained in step S3 in reverse phase C18 columns, first uses water-methanol gradient elution, so
Again with methanol-acetone gradient elution afterwards collects the fraction containing Aurantiamide acetate, and removal solvent is concentrated under reduced pressure.
2. according to the method described in claim 1, it is characterized in that, preferred, the Radix Isatidis is rhizoma et Radix Baphicacanthis Cusiae；
Preferably, silica gel described in step S3 is purification on normal-phase silica gel；
Preferably, in step S3 ethyl acetate-light petrol according to volume ratio 9:1~5:1 ratio carries out gradient elution；
Preferably, in step S4, first use water-methanol according to volume ratio 100:0~0:100 ratio carries out gradient elution, then
Again with methanol-acetone is according to volume ratio 100:0~50:50 ratio carries out gradient elution；
Preferably, further include step S5, the product that step S5 is obtained with water dissolution step S4 uses volume ratio for 70:30 second
Nitrile-water carries out liquid phase preparation as eluant, eluent, and the Aurantiamide acetate of purity >=90% is made.
3. application of the Aurantiamide acetate in the drug for preparing resisiting influenza virus.
4. application according to claim 3, which is characterized in that Aurantiamide acetate is preparing and influenza virus can inhibited to lure
It is applied in the drug for the inflammatory reaction led.
5. application according to claim 4, which is characterized in that Aurantiamide acetate can inhibit Flu-A sick in preparation
It is applied in the drug of the inflammatory reaction of poison induction.
6. application according to claim 3, which is characterized in that the Aurantiamide acetate is to detach to obtain from Radix Isatidis
?.
7. application according to claim 6, which is characterized in that the Radix Isatidis is rhizoma et Radix Baphicacanthis Cusiae.
8. application according to claim 3, which is characterized in that the Aurantiamide acetate is any according to claim 1-2
Method described in obtains；And/or pharmaceutically acceptable dosage form is made in the drug.
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