CN101829113B - Application of berberine in medicine for treating or preventing influenza virus - Google Patents

Abstract

The invention discloses an application of berberine in medicine for treating or preventing influenza virus. The application proves that berberine enjoys the good function of resisting influenza virus on MDCK cells infected by A-type influenza virus such as H5N1 and H3N2 and on A549 cells.Meanwhile, the berberine can kill influenza virus to a certain extent, prevent attack from the influenza virus and inhibit cells infected by the influenza virus; the berberine enjoys similar effect to amantadine, a positive control medicine; in addition, the berberine can well inhibit inflammatory responses triggered by the influenza virus and is disclosed to enjoy the prospect of being developed into the medicine for resisting the influenza virus.

Description

The application of berberine in treatment or flu-prevention virus drugs

Technical field

The present invention relates to medical technical field, more specifically relate to the application of berberine in treatment or flu-prevention virus drugs.

Background technology

Influenza virus is to cause grippal cause of disease, is divided into first (influenza A virus), second (influenzaB virus), third (influenza C virus) three types, and is wherein maximum to threat of human with influenza A.The human influenza virus mainly is H1, H2 and H3 hypotype, is mostly H5, H7 and H9 hypotype and endanger serious high pathogenic avian influenza virus at present, and promptly H5N1, H7N7, H9N2 etc. are wherein high with H5N1 hypotype case fatality rate.Therefore the present invention is that object of study is inquired into the antiviral activity of berberine with H5N1, two kinds of A types of H3N2 influenza virus mainly.

Influenza Virus orthomyxoviridae family (Orthomyxoviridae) has 8 fragments of peplos, and the strand of the 11 kinds of protein of encoding is altogether born RNA viruses.Different with other RNA viruses is that the synthetic of all RNA of influenza virus (mRNA, cRNA, vRNA) all accomplished in by the nuclear of infection cell.MRNA transfers to endochylema after in nuclear, synthesizing, the structure and the non-structural protein of synthetic virus; Then begin to assemble influenza virus, discharge progeny virion with the mode of sprouting after accomplishing, further invade new host cell.

In each gene of influenza virus, the NP gene is the most conservative.NP has type specificity, and still a kind of multifunctional protein except the nucleocapsid that forms virus, also can be stablized vRNA through RNP, makes it to avoid the effect of RNA enzyme.Research for many years thinks that PB1 is the catalytic subunit of viral rna polymerase, is responsible for duplicating and transcribing of viral RNA; PB2 is responsible for being used for virus mRNA with the CAP cap sequence that the mode of a kind of being called " Snatch " is captured host mRNA to transcribe.Can influenza virus effectively duplicate in human body cell, main relevant with rdrp virus (PB1, PB2 and PA) gene.

At present, influenza still belongs to and fails the effectively acute upper respiratory tract infectious disease of control.Because the antigenic variation ability of influenza virus is strong; The difference of individual immunity power in addition; The protective rate of vaccine is not high, and vaccine only has preventive effect to known influenza virus sub-strain, and invalid for the new type influenza virus that is produced by antigenic drift or antigenicity conversion.The medicine that current FDA approval is used to prevent and treat influenza has amantadine (Amantadine), rimantadine (Rimantadine) and zanamivir (Zanamivir), but has the problem that is prone to cause the appearance of influenza virus persister and takes inconvenience respectively.The Chinese medicine medicine comprises the Chinese patent medicine of heat-clearing and toxic substances removing such as QINGKAILING KOUFUYE, SHUANGHUANGLIAN ZHUSHEYE, HUOXIANG ZHENGQI WAN, SHENMAI ZHUSHEYE, enhancing human immune, and these medicines have advantages such as comprehensive therapeutic effect is good, toxic and side effects is low.Therefore, the research of anti-influenza virus medicament is in the ascendant.Chinese herbal medicine is natural treasure-house, and the effectiveness with its inherent science and practice receives countries in the world medicine and pharmacology worker's concern just day by day.Advantages such as Chinese medicine has not only that drug resistance is low, side effect and untoward reaction are few; And suppress virus replication in addition, regulate immunologic function, improve blood circulation, antipyretic-antalgic, and comprehensive effect such as anti-inflammation, it has special advantages and vast potential for future development aspect control influenza virus.Therefore, in recent years, all carry out the research of resisiting influenza virus Chinese herbal medicine both at home and abroad, obtained result preferably.

Rhizoma Coptidis is the dry rhizome of ranunculaceae plant Rhizoma Coptidis Coptis chinensis Franch., Coptis deltoidea C.Y.Cheng et Hsiao Coptis deltoideaC.Y.Cheng et Hsiao or Coptis Teeta Wall Coptis teeta Wall..Has heat clearing and damp drying, effects such as eliminating fire and detoxication.Be used for that damp and hot feeling of fullness, vomiting acid regurgitation, dysentery, jaundice, unconsciousness due to high fever, hyperactivity of heart-fire, dysphoria and insomnia, heat in blood are told nosebleed, conjunctival congestion, have a toothache, quenched one's thirst, the carbuncle furuncle; External treatment eczema, eczema, auditory meatus are suppurated.Berberine is a kind of important alkaloid, is that China uses Chinese medicine for a long time.Can from plants such as Rhizoma Coptidis, Cortex Phellodendri, Radix Berberidis, extract.It has significant bacteriostasis.The bibliographical information alkaloid compound has the effect of resisiting influenza virus in the recent period; Berberine is the main effective ingredient of Rhizoma Coptidis; The report that its resisiting influenza virus effect is not also arranged at present; Therefore the present invention has found that first berberine has good anti-influenza virus activity, shows that it has good exploitation prospect.

Summary of the invention

The object of the present invention is to provide the application of berberine in treatment or flu-prevention virus drugs, thereby be that influenza virus property treatment of diseases provides a kind of safe and effective toxic and side effects little natural drug clinically.

In order to realize above-mentioned task, the present invention adopts following technical scheme:

Berberine to the inhibitory action of influenza virus, shows that this medicine has the active drug that is developed to resisiting influenza virus and is applied to clinical on cell and molecular level.Through hemagglutinative titer test (HA) and the test of Real-Time quantitative fluorescent PCR, to the dosage and the effectiveness of the active drug of screening are made analysis in earlier stage.Adopt cell in vitro model---mdck cell simultaneously, from absorption and the invasion of directly killing the virus, suppress virus, the antiviral mechanism of duplicating, influence aspect research medicines such as viral membrane that suppresses virus.

Result of study shows that berberine has good antivirus action to influenza virus on cellular level, shows that this medicine can be used as potential clinically antiviral drugs and further develops.

The invention discloses a kind of berberine as the application in preparation treatment or the flu-prevention virus drugs, estimate in the face of the effect of berberine resisiting influenza virus from cell and molecular layer, for its further Application and Development is had laid a good foundation.

Because berberine has certain infection and inflammatory effect; Simultaneously be used for enteritis property disease clinically good application foundation is arranged; And often be attended by inflammatory reaction in the viral caused infectious disease generating process, so this medicine all has alleviation and auxiliary treatment effect preferably to symptoms such as the caused inflammation of virus in the treatment viral disease.

Berberine mainly is used for enteritis property disease with tablet clinically at present, in conjunction with modern common drug preparation means, can be made into thin membrane coated tablet, capsule, granule, dispersant, and is oral.In the medicine of treatment influenza infection and complication thereof, can add this medicine composition or this medicine of drug combination use in therapeutic process.

For other present anti-influenza virus medicaments, the present invention compared with prior art has the following advantages and effect: 1, this medicine is a kind of pure natural medical, has advantages such as toxic and side effects is little.2, all effective to first, second, influenza virus C.3, existing therapeutical effect also has preventive effect.4, Orally-administrable, easy to use.5, be non-nucleosides compound, have antiinflammatory action concurrently,, improve therapeutic quality and give drug compliance, alleviate the treatment misery so can improve the toleration in the therapeutic process.

The acquisition of A, berberine

Berberine (chemistry berberine hydrochloride by name, C ₂₀H ₁₈ClNO ₄.2H ₂O); Main middle separation acquisition from the dry rhizome of ranunculaceae plant Rhizoma Coptidis Coptis chinensis Franch., Coptis deltoidea C.Y.Cheng et Hsiao Coptis deltoidea C.Y.Cheng et Hsiao or Coptis Teeta Wall Coptis teeta Wall.; Adopt alkaloid extraction separation and purification means and technology path in " Chinese medicinal plant chemistry ", its purity detects through HPLC to be had more than 98%.

The efficacy study summary of B, berberine

Berberine is mainly derived from the medicinal plants Rhizoma Coptidis, and this plant is a heat-clearing and toxic substances removing good medicine very important simply in traditional Chinese medicine.

Berberine is used for treatment diarrhoea as nonprescription drugs always in clinical; Have significant heart failure resistance, arrhythmia, cholesterol reducing, inhibition vascular smooth muscle propagation, improve effects such as insulin resistant, antiplatelet, antiinflammatory but modern pharmacology research confirms berberine; Thereby unify at cardiovascular system will have aspect the nervous system disease extensively, important application prospects, come into one's own day by day.

Berberine is a kind of important alkaloid, is that China uses Chinese medicine for a long time.Can from plants such as Rhizoma Coptidis, Cortex Phellodendri, Radix Berberidis, extract.It has significant bacteriostasis, is commonly used to treat digestive tract disease such as bacillary gastroenteritis, dysentery.Clinical being mainly used in treated bacillary dysentery and gastroenteritis, its no Drug resistance and side effect.

And also do not appearing in the newspapers and application aspect the influenza virus disease therapeutic about it.So the present invention is that the treatment of the susceptible viral disease of convection current provides a kind of new active drug to the discovery of its new drug with value.

The present invention compared with prior art has the following advantages and effect:

1, berberine does not have cytotoxicity in big concentration range.

Adopt H5N1 to infect influenza virus easy infection cells such as mdck cell or A549, in virocyte gets into cyton after, add the berberine of variable concentrations, the OD value of influenza virus infection cell before and after the inspection berberine adds, the survival rate of calculating cell,

Table 1 berberine is to mdck cell toxicity test result

Can find out that by table 1 berberine pair cell in bigger concentration range does not have toxicity, shows that the safety of medicine is better.

2, berberine can improve the preventive effect of cell to viral infection

Adopt hemagglutinative titer experiment (HA) detection of drugs to the suppression ratio of virus and the binding mode of medicine; Carry out the mechanism of action of medicine and tentatively inquire into, from medicine to the absorption of virus and invasion, virus intravitally duplicate at cell, medicine infects the preliminary discussion that equal angles has been carried out the effect characteristics to lethal effect, the drug influence cell resisiting influenza virus of virus

The influence that table 2 different modes of administration is duplicated influenza virus

It is similar that the result shows that berberine and virus join the exercising result that adds medicine behind inhibition virus function effect and the virus infected cell among the cell again after hatching jointly, shows that berberine can not direct killing virus, but can suppress viral duplicating.Medicine joins after the cell experimental result that virus is joined among the cell again and shows that its action effect and other administering mode that suppresses virus does not have notable difference property yet; Explain that medicine can improve the resistant function of cell to virus, but can not stop the invasion of virus.

3, berberine can suppress the inflammatory reaction that viral infection causes

Adopt the PGE2 detection kit, the A549 cell conditioned medium liquid 36, after collecting viral infection during 48h is measured the PGE2 content in the cell conditioned medium according to the kit measurement method respectively.

Table 3 medicine is to the influence of PGE2 in the influenza infection A549 cell conditioned medium liquid

The result show virus infected cell after 48 hours inflammatory reaction apparent in view, berberine obviously reduces the content of PGE2 after using 48 hours, show the inflammatory reaction that medicine is caused in can low virus infection, play the function of protection body.

Description of drawings

Fig. 1 drug cell toxicity test, variable concentrations medicine adopt MTT to observe the influence of medicine cell growth after joining and cultivating 24h in the cell.

After Fig. 2 berberine joins when 100 μ g/ml concentration and cultivates 24h in the mdck cell, the variation of observation of cell form under the ultramicroscope.A does not have the drug treating group; B adds the berberine processed group.

Fig. 3 variable concentrations medicine joins when cultivating 48h after the virus infected cell, adopts MTT to measure the cell survival rate situation.

Fig. 4 hemagglutinative titer test (48h) variable concentrations medicine is to the inhibitory action of virus multiplication.

Fig. 5 different drug joins in the mdck cell of infective virus, tests the virus titer that detects in the different time points cell conditioned medium through HA.

Fig. 6 adopts the method for plaque ethods (plaque reduce assay) to detect the inhibitory action of berberine to influenza virus.(the Testis et Pentis Canis cell monolayer infected with influenza A virus that goes down to posterity through absorption in 1 hour, is removed cell conditioned medium liquid; And adding 2ml pastille culture medium (drug level is 100ug/ml); Cultivate after 24 hours, get cell conditioned medium liquid, collect 150ul cell conditioned medium liquid and join in another 6 orifice plates that cover with the monolayer mdck cell; After 1 hour, in the cell that infects, cover the solid medium that contains 0.5% agar 37 ℃ of effects then.Hatch back 3 days at 37 ℃, 10% formalin film is fixed, dyeing, 1% violet staining.Blue dyeing shows that cell does not receive the virus influence; The result of being unstained and plaque occurring shows that cell receives the erosion of influenza virus.

Fig. 7. add the mdck cell of the infection H5N1 of different pharmaceutical, carry out immunofluorescence test.A. the negative control of uninfecting virus; B. the cell of infective virus contrast; C. be the experimental group that adds berberine behind the infective virus; D. be the experimental group that adds amantadine behind the infective virus.

Fig. 8 adopts the influence of real-time PCR detection of drugs to influenza virus reproduction process virus load.A.NP specific fragment mRNA (* p＜0.05; * p＜0.01); B.NP specific fragment cRNA (* p＜0.05; * p); C.NP specific fragment vRNA (* p＜0.05; * p＜0.01).

Medicine is to the inhibitory action of influenza virus during Fig. 9 .1 different modes of administration.At 24 hours collecting cell supernatant of drug effect, adopt HA to detect virus titer.Data are mean+SD (3 of each concentration are parallel).With model group relatively, * p＜0.05 (difference of virus titer surpass 2 times can be considered apparent property difference is arranged), medicine can not influence the absorption and the invasion of virus as a result.

Medicine is to the inhibitory action of influenza virus during Fig. 9 .2 different modes of administration.At 36 hours collecting cell supernatant of drug effect, adopt HA to detect virus titer.Data are mean+SD (3 of each concentration are parallel).With model group relatively, * p＜0.05 (difference of virus titer surpass 2 times can be considered apparent property difference is arranged), medicine can not influence the absorption and the invasion of virus as a result.

Medicine is to the inhibitory action of influenza virus during Fig. 9 .3 different modes of administration.At drug effect collecting cell supernatant after 48 hours, adopt HA to detect virus titer.Data are mean+SD (3 of each concentration are parallel).With model group relatively, * p＜0.05 (difference of virus titer surpass 2 times can be considered apparent property difference is arranged), medicine can not influence the absorption and the invasion of virus as a result.

Interaction experiment between Figure 10 .1 mammal double cross test detection assay NP and the berberine.In the HeLa cell, transfection reporter plasmid pG5Luc carries the NP gene with test plasmid pvP16 nucleoprotein (NP) respectively, behind 6h, adds medicine then respectively.The uciferase activity of NP and drug interaction is measured.Negative contrast of plasmid pVP16 (VP16) and background control contrast.Data are mean+SD (3 parallel appearance).Compare * p＜0.05 with positive control; * p＜0.01 expression has significant difference.

Figure 10 .2 mammal double cross test detects berberine and whether suppresses the interaction between NP and the NP.

Figure 10 .3 animal double cross test detects the berberine element and whether suppresses NP, PB2 interaction between protein

Figure 10 .4 breast animal double cross test detects Rhizoma Coptidis

Figure 11 is whether an ethnic Shi Tu suppresses NP, PB1 interaction between protein

Figure 12 is a process chart.

The specific embodiment

The application of a kind of berberine in treatment or flu-prevention virus drugs, its application process is:

At present normal tetramethyl azo azoles salt (MTT) method that adopts, detection of drugs to influenza infection after the diversity of mdck cell survival rate carry out drug screening.Also there are some to be directed against the medicine in-vitro screening that key enzyme that influenza virus duplicates carries out in addition; But Comparatively speaking; Employing cell culture method for screening can be simpler, and can system inquire into concentration medicine and therapeutic index, for more basic later stage mechanism research provides.Process chart is asked for an interview Figure 12.

Embodiment 1: the evaluation of berberine anti-influenza virus activity

1, material: cell, virus and plasmid:

Mdck cell is provided by Chinese Academy of Sciences's Shandong medical microbial institute, and A549 cell, HeLa cell, viral H5N1 or H3N2 purchase in Chinese representative microbial preservation center; PVP-16-NP, pM-PB1, pM-PB2, pVP-16-NP, pG5Luc plasmid instruct completion by the Wu Jianguo professor of medical virology seminar of virusology National Key Laboratory of Wuhan University, and pG5Luc is the reporter gene carrier that has green fluorescence GFP, and plasmid pVP-16-NP, pM-PB1, pM-PB2, pVP-16-NP are respectively that the different albumen of influenza virus are connected on carrier pVP and the pM, paper---the Mukhtar that visible this laboratory of relevant details has been delivered; M.M., Li, S., Li; W., Wan, T., Mu; Y., Wei, W., Kang; L., Rasool, S.T., Xiao; Y., Zhu, Y.; And Wu, J. (2009) .Single-chain intracellular antibodies inhibit influenzavirus replication by disrupting interaction of proteins involved in viral replication andtranscription.Int JBiochem Cell Biol 41 (3), 554-60.)

2, toolenzyme:

LA-taq, LC-taq, M-MLV Reverse Transcriptase, M-MLV RT 5X Buffer, RNAexhibit is available from precious biological (TaKara) company;

3, other reagent:

The DMEM powder is available from Gibco company; Sofast ^TMTransfection reagent is available from Shanghai sun horse company; Survey fluorescent instrument: Turner BioSystems TD-20/20 fluorophotometer (Turner Designs, Sunnyvale, CA); TRIzol reagent is available from Gibco company; Berberine (Berberine) is self-control, and it is more than 98% that HPLC detects purity.

Drug toxicity detects test

At first different pharmaceutical concentration is estimated mdck cell toxicity.

1, mdck cell in the digestion T25 bottle adds an amount of cell culture medium, piping and druming cell 10-15 time.

2, cell is taped against in 96 orifice plates, is positioned in the incubator, it is for use to cultivate 24h.

3, the mdck cell of cultivating is taken out, remove original culture fluid, use the gradient medicine of keeping the culture medium dilution of 2%FBS instead, it is for use to cultivate 24h.

4, the medicine that adds variable concentrations then after continuing to cultivate 48h, is abandoned the culture fluid supernatant, adds 5mg/mL MTT liquid 50 μ L/ holes (the DMEM culture medium not contain serum is mixed with) in culture plate, puts and continues in the CO2 incubator to cultivate.

5, behind the cultivation 2-3h, abandon the MTT supernatant, PBS washes 3 times, and every hole adds lysate (DMSO: the ethanol volume ratio is 1: 1) 100 μ L, vibrates 5-10 minute.

6, dissolving fully to be crystallized is put on the enzyme mark analyzer, measures the optical density OD value of 570nm (490nm) wavelength.

7, find out the maximal non-toxic concentration range of medicine pair cell.

The result shows that medicine is at 0.16ug/ml---the scope of 500ug/ml does not have obvious cytotoxicity (Fig. 1 .) to this cell, and examining under a microscope cell does not have pathological changes generation (Fig. 2 .) yet, therefore explains that medicine has the scope of application of comparison safety.

The screening test of the anti-H5N1 virus of berberine

Whether A, HA detection of drugs have the resisiting influenza virus effect

1, with pancreatin well-grown mdck cell is dispersed into the individual cells suspension, by 1 * 10 ⁵/ mL concentration branch is added on 48 orifice plates, every hole 0.2mL.

2, put in 37 ℃, 5%CO2 incubator and cultivate 24h.The mdck cell of cultivating is taken out, remove original culture fluid, change the nonresistant culture medium of serum-free, add the viral liquid of 1M.O.I, hatched 1-2 hour in 37 ℃.

3, pour out viral liquid, use the gradient medicine of keeping the culture medium dilution of 2%FBS instead.If blank, positive control, negative control.The experimental group medicine has six gradients.

4, put 37 ℃, 5%CO ₂Cultivate in the incubator, collect each experimental port virus supernatant behind the 36h and carry out the blood clotting experiment.

5, select 96 hole blood clotting brassboards for use, except that first hole, every hole adds the PBS of 75 μ L.

6, first hole adds 75 μ L viral suspension to be measured, and proportional diluted discards 75 μ L during to the 12nd hole then.

7, every hole adds 1% chicken erythrocyte suspension, 75 μ L, flicks brassboard, and erythrocyte is fully mixed with virus, and room temperature (identical below 20-25 ℃) is observed the blood clotting phenomenon and write down the result when leaving standstill 30min, 45min, 60min.

8, the blood clotting phenomenon when leaving standstill 45min is a final result.With the high dilution of the influenza virus that occurs " ++ " (be that erythrocyte forms a ring-type at the bottom of the hole, little coagulation piece is arranged) all around is the blood clotting terminal point, and this dilution inverse is the blood clotting titre of influenza virus.Normal saline contrast (nonspecific agglutination of getting rid of hemocyte self) and virus control are established in experiment simultaneously.

9, Processing Test data draw the antiviral optium concentration of each medicine of test group.

10, by above-mentioned result of the test, get the antiviral optium concentration of each medicine and make an experiment.

11, repeat the step of above-mentioned 1-9, at this moment, only add the optium concentration (need not to do the gradient test) of antiviral drugs, get the temporal correlation test that supernatant is done virus respectively at 24h, 36h, 48h.

On the basis of the above; Whether the administration of having investigated various dose shows different effects to influenza virus; The result shows does not have tangible dosage according to patience relation (Fig. 3 .) yet when berberine acts on influenza virus; Possible cause is in the drug use safe concentration scope, and medicine has reached the effect of better inhibited virus replication than low dosage the time, and along with the increase of drug level; Significant change can't take place in its action effect, also points out this medicine dosage when treatment suitably to adjust.

Detected the virus titer in the cell conditioned medium liquid behind the drug treating viral infection simultaneously; The result shows that equally the action effect of different pharmaceutical is closer like (Fig. 4); Though there is not the dose dependent relation; But the cytotoxicity that medicine also is described is lower, and medicine can be brought into play antivirus action in low concentration and big concentration range.

According to above-mentioned experimental result, select for use the cell administration concentration of berberine and amantadine to be 100ug/ml, inquired into different effects virus titer in the virus infected cell supernatant after the time; Count with the viral dilution multiple, the result is shown in Figure 5, and different effects time point experimental result shows that virus titer is along with the prolongation of viral infection time constantly increases; Reach peak at 48h; And berberine and positive control medicine amantadine just show good inhibition virus effect when 24h, and along with the prolongation of action time, its effect that suppresses virus is also strengthening; Show that medicine can suppress duplicating of virus, and can keep action time.

B, plaque ethods detect the effect whether berberine has resisiting influenza virus

Experimentation: behind the virus infected cell 1h, add different pharmaceutical then respectively, behind drug effect 24h; Collecting cell supernatant 150ul joins six orifice plates that cover with cell monolayer respectively, after viral supernatant absorption 1h; Add 0.5% agarose according to traditional plaque ethods, continue then to cultivate 72h, after 0.4% the formaldehyde fixed; Adopt violet staining, plaque situation behind the observation drug effect.

The result shows that berberine has reasonable antiviral and duplicates effect, and its cell of adding berberine is more complete in the cell of viral infection, almost can't see viral plaque, and its action effect is similar to positive control drug amantadine (Fig. 6).

C, immunofluorescence test detect virus infected cell

1, immunofluorescence dyeing operation

1, the preparation of fixative: as fixative, or use the fixative of effective ingredient as ethanol, methanol or other type according to specific one anti-or sample with 4% paraformaldehyde.

2, the preparation of cleaning mixture:

The preparation of 10XTBS: take by weighing 24.2g Tris (Tris alkali), 80g NaCl; Regulate pH value to 7.6 with hydrochloric acid, be settled to one liter.TBS preparation: be diluted with water to 1X to 10XTBS according to 1: 9 ratio and be TBS.

The preparation of TBSTx: in TBS, adding Triton X-100, to make ultimate density be 0.1%, and mixing is TBSTx.

3, the preparation of confining liquid:

The TBSTx that preparation contains 5%BSA is as confining liquid:

Contain the preparation of the TBSTx of 5%BSA: in 100 milliliters of TBSTx, add 5 gram BSA, dissolving and mixing are the TBSTx that contains 5%BSA.

4, the preparation of an anti-diluent:

Use above-mentioned confining liquid, promptly contain the TBSTx of 5%BSA, as an anti-diluent.

One anti-dilution: one anti-ly is mouse-anti people H5N1 HA monoclonal antibody.Resist with an anti-diluted one according to the dilution ratio that is used for immunofluorescence dyeing of recommending in the anti-description.

5, the anti-dilution of fluorescent labeling two:

Fluorescently-labeled two anti-, dilute with the immunofluorescence dyeing two anti-diluents that this test kit provides according to 1: 1000 ratio.According to the power of fluorescence, the ratio of dilution can suitably improve or reduce.

Under fluorescence microscope, its result is as shown in Figure 7.Negative control group does not produce fluorescence under exciting light; Viral infection does not add any drug treating matched group and under green fluorescence excitation, produces much more very red fluorescences in the visual field, shows that the HA protein content of expressing in the cell is very big, proves by the mdck cell of influenza infection a lot; It is similar with the viral infection matched group with the experimental group fluorescence result of polypeptide 165 to add medicine UA, explains that these two kinds of medicines do not have tangible antivirus action; The experimental group cell that adds the medicine berberine, the few and fluorescence of the fluorescence that is inspired a little less than, visiblely infected H5N1 virus and expressed the proteic cell of HA less, explain that berberine has antivirus action very significantly; The fluorescent effect and the berberine of positive controls amantadine are close.

D, Real-Time RT-PCR detect the influence of berberine to virus replication

For detection of drugs to the virus transcription and the influence of duplicating, detected three kinds of multi-form viral RNAs of A type influenza virus (mRNA, cRNA, vRNA) through the real time fluorescent quantitative test.Different pharmaceutical adds the mdck cell that infects H5N1 virus, receives appearance after 4 hours and extracts RNA.With different primers mRNA, cRNA, the vRNA reverse transcription of virus become cDNA,, detect the amount of virus mRNA, cRNA, vRNA again through the fluorescent quantitation test.

Design of primers

1, RT-PCR primer

mRNA：5’-TTTTTTTTTTTTTTTTTT-3’

NP-cRNA：5’-AGTAGAAACAAGGGTATTTTTCTTTAATTGTCAT-3’

NP-vRNA：5’-CTCACCGAGTGACATCAACATCATG-3’

2, Real-Time PCR primer

NP：sense 5’-GGATTTGGCGTCAAGCGAACA-3’

antisense 5’-GTCCCTACCCCCTTTACTGC-3’

г-actin：sense 5’-TCTGTCAGGGTTGGAAAGTC-3’

antisense?5’-AAATGCAAACCGCTTCCAAC-3’

The extraction of viral RNA

1, cultivates mdck cell with 6 orifice plates, add the medicine that adds optium concentration behind the viral 1.5h respectively, establish blank, positive control and negative control;

2, every hole adds 1ml Trizol homogenate behind the 4h;

3, move into the new centrifuge tube of 1.5ml;

4, hatch 5 minutes on ice;

5, centrifugal 12, get supernatant in 5 minutes for 000rpm4 ℃ and move into the new centrifuge tube of 1.5ml;

6, add the 0.2ml chloroform, concussion was hatched 5 minutes on ice;

7,12,4 ℃ of 000rpm got upper strata liquid in centrifugal 10 minutes and move into the new centrifuge tube of 1.5ml;

8, add the 0.5ml isopropyl alcohol, concussion was hatched 5 minutes on ice;

9,12,000rpm4 ℃ of centrifugal 5 minutes abandoning supernatant;

10, add 1ml 75% ethanol, concussion;

11, centrifugal 7,4 ℃ of 5 minutes abandoning supernatant of 500rpm;

12, make it bleach under the room temperature;

13, add DEPC treating water 10 μ l-35 μ l dissolving RNA (can be kept at liquid nitrogen or cryogenic refrigerator).

The RT-PCR reaction system

The first step:

RNA extract 2 μ l,

Each 1 μ l of 20mM primer,

70 ℃ 10 minutes, put into ice bath immediately.

Second step:

5x PT-PCR buffer 5 μ l,

RNA?sin 0.5μl，

25mM?MgCl2 1.5μl，

2.5mM?dNTP 2μl，

AMV reverse transcriptase 1 μ l,

Sterilized water 11 μ l;

37 ℃ 1.5 hours, 94 ℃ of 10min.

Real-Time PCR reaction system

Each 1ul of upstream and downstream primer

10xPCR buffer 2.5 μ l,

25mM?MgCl2 1.5μl，

Dna profiling 1 μ l,

SYBR?green?I 1μl，

2.5mM?dNTP 2μl，

Taq enzyme 1 μ l,

Sterilized water 14 μ l;

Reaction condition: 1,95 ℃ 5 minutes, be first step 1 circulation; 2,95 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute was second step 40 circulations; 3,72 ℃ 10 minutes.

The result of Real-Time PCR shows: with respect to matched group, the mRNA of virus N P specific fragment significantly reduces (Fig. 8) under the effect of amantadine (p＜0.01), berberine (p＜0.01) and UA (p＜0.05).The cRNA of virus N P specific fragment can be suppressed by berberine (p＜0.01) and amantadine (p＜0.01) significantly, and the action effect of berberine will be higher than amantadine; With respect to matched group, polypeptide 165 (p＜0.05) also has very strong inhibitory action (Fig. 8) with formation to the cRNA of NP.And the segmental vRNA of virus N P only, just can significantly reduce when existing at amantadine (p＜0.01);

Embodiment 2: berberine prophylaxis of viral infections, to influenza virus absorption invasion, the influence of duplicating

The cell that will be paved into monolayer respectively carries out different processing modes: medicine joins in advance hatches in the cell after the 1h, changes liquid, adds corresponding influenza virus; Medicine joins influenza virus absorption invasion employing medicine and hatches 1h in the virus jointly, and then joins in the cell; Medicine is to the influence of duplicating of influenza virus, adopt viral first infection cell 1h after, add medicine then.The drug level that uses in all experiments is 100ug/ml, and the viral infection amount is the viral liquid of 1M.O.I.Respectively 24,36, collecting part supernatant during 48h, adopt HA to measure the antivirus action of medicine.

The result is as shown in Figure 9, and berberine does not influence the absorption and the invasion of virus, but to virus to duplicate inhibitory action apparent in view, medicine has certain preventive effect simultaneously, can improve the resistant function of cell to viral infection.

Embodiment 3: the influence of medicine to some enzymes in the reproduction process inquired in nurture animal double cross test

1, cell transfecting

Plasmid is respectively pM-PB1, pM-PB2, pVP-16-NP, pG5Luc.This test group is established negative control and is not added any plasmid, and positive control adds pM-PB1 (or pM-PB2), pVP-16-NP, three kinds of plasmids of pG5Luc, and experimental group adds pM-PB1 (or pM-PB2), pVP-16-NP, three kinds of plasmids of pG5Luc and various medicine.

Sofast ^TMLiposome transfection

Following transfection operation is an example with 24 orifice plates.The transfection overall process must the Strict aseptic operation.

1. will treat that transfectional cell is inoculated in 24 orifice plates, 37 ℃ of cultivations the previous day in transfection.Cell density before the transfection is as the criterion with 40-60%.

2. prepare following solution

1. dilute 1.5 μ lSofast ^TMReagent is in the DMEM culture medium of 30 μ l serum-frees, no antibiotics.Room temperature left standstill 3 minutes behind the mixing.

2. dilute 0.6 μ g and treat the DMEM culture medium of transfection DNA in 30 μ l serum-frees, no antibiotics.Room temperature leaves standstill behind the mixing.

3. mix 1., 2. two kinds of solution, room temperature left standstill 20 minutes.

4. discard the old culture medium in the culture plate, add the DMEM culture medium of 0.5ml serum-free, no antibiotics.

5. add above-mentioned Sofast ^TMReagent-DNA mixture is in 24 orifice plates.

6.37 change 10% serum and 1% antibiotic DMEM culture medium after ℃ incubation 4-6 hour.

7.24 after change serum-free DMEM culture medium, 37 ℃ of cultivations.

8.48-72 detect after hour.

2, uciferase activity is measured

1, takes out luciferase substrate and luciferase lysate balance at room temperature.

2, with PBS rinsing cell.This process must be careful, do not contact attached cell.At last as far as possible with the PBS sucking-off.

3, add enough lysis buffers to cover cell.For example: 400 μ l/60mm culture dishs, the every hole 20 μ l of 900 μ l/100mm culture dishs or 96 orifice plates.Rock culture dish for several times to guarantee that lysis buffer covers cell fully.

4, cell is scraped from culture dish.Cell and all liquid are transferred in the centrifuge tube.Centrifuge tube is placed on ice.Vortex shakes centrifuge tube 5-10 second, then with centrifugal 15 seconds of 12000g (room temperature), supernatant is transferred in the new test tube.

5, getting about 30 μ l (used lysate total amount is 100 μ l) mixes in the Ep of 1.5ml pipe with the good substrate of the balance of 50 μ l; Then immediately at Turner BioSystems TD-20/20 fluorophotometer (TurnerDesigns; Sunnyvale, CA) activity of the luciferase of last test sample.

Berberine (Berberine) can not suppress the absorption invasion of virus.So through mammal double cross test, detect berberine whether the nucleoprotein (NP) through suppressing virus thus influence viral duplicating with interaction between PB1, the PB2.

Result of the test is shown in Figure 10 .1: each experimental group medicine does not have very remarkable influence (Figure 10 .2) to NP, the interphase interaction of PB1 albumen; And berberine can suppress NP, PB2 interaction between protein, and with respect to positive control, suppression ratio reaches 50% (Figure 10 .3).This experimental result shows that berberine possibly be to suppress duplicating of virus through the combination that reduces NP, PB2.

Berberine is to the influence of the caused inflammatory reaction of influenza infection

Adopt the PGE2 detection kit, the A549 cell conditioned medium liquid 36, after collecting viral infection during 48h is measured the PGE2 content in the cell conditioned medium according to the kit measurement method respectively.The result is shown in figure 11; The PGE2 that virus infected cell produced in the time of 36 hours does not have significant change; And content is very low; When measuring in 48 hours Deng virus infected cell, the groups of cells PGE2 content that viral infection does not add any drug treating is the highest, with normal cell matched group and medication group significant difference (* p＜0.05 is arranged; * p＜0.01), berberine and positive control drug amantadine can significantly reduce PGE2 secretion (with infection group and viral infection group relatively, * p＜0.05; * p＜0.01).Explain that berberine can significantly reduce the secretion of some inflammatory factors that cause in the virus infection.

Claims (1)

1. the application of berberine in preparation treatment or H5N1 infection of prevention influenza A virus and inflammation medicine thereof.

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