CN109674780A

CN109674780A - Quercetin and its derivative are preparing the application in anti-zika virus drug

Info

Publication number: CN109674780A
Application number: CN201910047562.4A
Authority: CN
Inventors: 邹敏; 柳红妙; 姚新刚; 周春琼; 柯昌文; 杨海涛; 刘叔文
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2019-01-18
Filing date: 2019-01-18
Publication date: 2019-04-26

Abstract

The invention discloses Quercetins and its derivative to prepare the application in anti-zika virus drug.Tests prove that: Quercetin has apparent anti-zika virus activity, it can obviously inhibit to infect plaque effect, mRNA and the protein expression level of zika virus generation and the activity of NS2B-NS3 protease, it acts on zika virus and enters the stage, can achieve 100% inhibitory effect.Quercetin is smaller to the toxicity of cell, to Vero cell substantially without toxicity in 0~12.5 μM of concentration range.IC of the Quercetin to zika virus₅₀It is 2.29 ± 0.55 μM.The present invention develops the new application of Quercetin, improves the economic value of Quercetin, and also for Quercetin, prepare the application in anti-zika virus drug, to provide theory and practice basic.

Description

Quercetin and its derivative are preparing the application in anti-zika virus drug

Technical field

The present invention relates to the applications of Quercetin and its derivative more particularly to Quercetin and its derivative to prepare anti-stockaded village's card Application in virus drugs.

Background technique

Zika virus is a kind of mosquito matchmaker transmitted virus, is just found early in nineteen forty-seven, and hereafter tranquillization is for many years, primary earliest quick-fried Hair prevalence is to occur for 2007 on the island Western Pacific Mi Keluoni subgroup Dao Yapu, and bigger is once popular in 2013-2014 Occur in year to have infected about 32000 people in Oceanian French Polynesia.Cause whole world extensive concern is 2015 Brazilian epidemic situation, the reason is that there is the newborn of thousands of microcephalus.Although the clinical symptoms of zika virus disease are generally relatively light, But since abnormal development of fetus can be caused, in addition it is related to actue infectious polyradiculoneuritis, thus cause to seriously affect, for this purpose, WHO announced that zika virus disease is the public health emergency of international concern on 2 1st, 2016.Although scientists are fast Horse studies relevant diagnostic kit, vaccine and drug with adding whip, but there is presently no zika virus vaccines and effective treatment Drug listing.

Zika virus category flaviviridae Flavivirus, spherical in shape, diameter is about 40~70nm, there is coating.Genome is single Stock positive chain RNA, length about 10.8kb have an open reading frame, encode a polyprotein containing 3419 amino acid. The protein cleavage is at three kinds of structural proteins, it may be assumed that capsid protein (C), memebrane protein precursor/memebrane protein (prM/M), envelope protein (E), And 7 kinds of non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5).3 kinds of structural proteins participate in the dress of virus Match, C protein is bonded the core of virion with geneome RNA；E protein mediates and combination and and the cell of host cell receptor The fusion of film plays a role during cell entry host cell；PrM assists the folding of E protein as a kind of chaperone Folded, to prevent virion from merging too early, prM is the precursor of albumen M, is cracked into albumen M to promote the maturation of virus.Non- In structural proteins, NS3 has serine protease, nuclear nucleoside triphosphatase and RNA helicase activity, wherein protease domain Play a part of to cut polyprotein precursor, form mature virus protein, helicase domain forms double-stranded RNA in virus replication After play despiralization, NS2B then combines NS3 to form stable compound, due to the compound in virus replicative cycle have it is more Weight function, NS2B-NS3 albumen can be used as the potential target spot of antiviral drugs, and grind for the anti-zika virus drug of the target spot Hair is still in infancy.

Vast territory and abundant resources in China, and be richly stored with natural pharmaceutical resources, has with natural drug treatment disease long Historical records provide substance and theoretical basis for exploitation antiviral natural drug.Quercetin is the one kind extracted from the sophora bud Flavone compound has lowering blood pressure and blood fat, norcholesterol, immunosupress, antiallergy, relieving cough and asthma, antitumor etc. a variety of Effect, but it has no relevant report in anti-zika virus drug.

Summary of the invention

The purpose of the present invention is to provide Quercetins and its derivative to prepare the application in anti-zika virus drug

The technical solution used in the present invention is:

One of the objects of the present invention is to provide Quercetins and its derivative to prepare the application in anti-zika virus drug.

Preferably, the derivative of above-mentioned Quercetin is its tautomer or its pharmaceutically acceptable salt.

Preferably, above-mentioned Quercetin pharmaceutically acceptable salt is its base addition salts, is selected from sodium, potassium, calcium, magnesium, iron, Asia Iron, ammonium or zinc salt；Or acid-addition salts, it is selected from sulfate, acetate, hydrochloride, phosphate, oxalates, maleate, fumaric acid Salt, malate, tartrate, citrate or benzoate.

Above-mentioned pharmaceutically acceptable salt can be easily separated, can be used conventional separation methods purification, as solvent extraction, dilution, Recrystallization, column chromatography and prepare thin-layer chromatography etc..

Preferably, above-mentioned zika virus is Chinese pathogenic strain Z16006 GenBank:955589.1.

It preferably, further include pharmaceutically acceptable carrier, diluent, excipient in above-mentioned anti-zika virus drug.

Preferably, the compound of the present invention can be made with pharmaceutically various typical additives (such as diluent and excipient) Pharmaceutical composition.According to therapeutic purposes and usage mode, pharmaceutical composition can be made to various types of administration unit dosage forms, such as Oral agents, sustained release agent, tablet, pill, pulvis, granule, capsule, oral agents, suppository and injection (solution and suspension) etc..

In order to shape the pharmaceutical composition of tablet form, it can be used this field any of and widely used figuration Agent.For example, carrier, such as lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, avicel cellulose and silicon Acid etc.；Adhesive, such as water, ethyl alcohol, propyl alcohol, common syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose Element, lac, methylcellulose and potassium phosphate, polyvinylpyrrolidone etc.；Disintegrating agent, such as dried starch, mosanom, agar powder and sea Band powder, sodium bicarbonate, calcium carbonate, the aliphatic ester of polyethylene sorbitan, lauryl sodium sulfate, stearic acid monoglycerides, Starch and lactose etc.；Disintegration inhibitor, such as white sugar, glycerol tristearate, coconut oil and hydrogenated oil and fat；Adsorption enhancer, such as Quaternary amine alkali and lauryl sodium sulfate etc.；Wetting agent, such as glycerol, starch；Adsorbent, such as starch, lactose, kaolin, swelling Soil and colloid silicic acid etc.；And lubricant, such as pure talcum, stearate, boric acid powder and polyethylene glycol etc..If necessary Words can also use common coated material to make tablet as sugar coated tablet, enteric coated tablets, film coated tablets (as applied gelatin diaphragm Agent), duplicature tablet and multilayer tablet.

In order to shape the pharmaceutical composition of pill, it can be used this field any of and widely used figuration Agent, for example, carrier, such as lactose, starch, coconut oil, hardened vegetable oils, kaolin and talcum etc.；Adhesive, such as gum arabic Powder, tragacanth gum powder, gelatin and ethyl alcohol etc.；Disintegrating agent, such as agar and Kelp Powder.

In order to shape the pharmaceutical composition of suppository form, it can be used this field any known and widely used figuration Agent, for example, polyethylene glycol, coconut oil, higher alcohol, the ester of higher alcohol, gelatin and semi-synthetic glyceride etc..

, can be by solution and suspension liquid disinfectant in order to prepare the pharmaceutical composition of injection form, and it is preferably added suitable chlorine Change sodium, glucose or glycerol etc. are made and the isotonic injection of blood.When preparing injection, it is possible to use in the art any Common carrier.For example, water, ethyl alcohol, propylene glycol, the isooctadecanol of ethoxylation, the isooctadecanol and polyethylene of polyoxy The aliphatic ester etc. of anhydro sorbitol.In addition, common lytic agent, buffer and analgesic etc. can also be added.

Another object of the present invention is to provide a kind of drug of anti-zika virus, the drug include Quercetin and/or Its derivative.

The beneficial effects of the present invention are:

1, tests prove that: Quercetin has apparent anti-zika virus activity, can significantly inhibit infection zika virus and produce The activity of raw plaque effect, mRNA and protein expression level and NS2B-NS3 protease, acts on zika virus and enters rank Section, can achieve 100% inhibitory effect.

2, Quercetin is smaller to the toxicity of cell, does not have poison substantially to Vero cell in 0~12.5 μM of concentration range Property.

3, IC of the Quercetin to zika virus₅₀It is 2.29 ± 0.55 μM.

4, the new application of present invention exploitation Quercetin, improves the economic value of Quercetin, also anti-in preparation for Quercetin Application in zika virus drug provides theory and practice basis.

Detailed description of the invention

Fig. 1 is toxicity detection figure of the Quercetin to Vero cell；

Fig. 2 is the flow chart of drug-treated and virus infection in Vero cell；

Fig. 3: (A) is the cell plaque effect figure of different dosing methods；It (B) is the zika virus of different dosing methods MRNA expression figure；

Fig. 4 is the opposite mRNA expression of the time that Quercetin is added after Vero cell is mixed with zika virus and zika virus Horizontal relational graph；

Fig. 5: the plaque effect figure of (A) Quercetin and zika virus incubation different time；(B) Quercetin is incubated with zika virus Educate the relational graph of time Yu zika virus NS1 protein expression level；

Fig. 6 is the relational graph of quercetin concentration and zika virus NS1 albumen inhibiting rate；

Fig. 7: (A) is NS2B-NS3 Proteinase bands figure；(B) it is changed over time for the fluorescence intensity of Different treatments Figure；It (C) is the NS2B-NS3 albumen enzyme inhibition rate relational graph of quercetin concentration and zika virus.

Specific embodiment

Enumerate embodiment further below with the present invention will be described in detail.It will similarly be understood that following embodiment is served only for this Invention is further described, and should not be understood as limiting the scope of the invention, those skilled in the art are according to the present invention Some nonessential modifications and adaptations that the principle of elaboration is made all belong to the scope of protection of the present invention.Following specific works of example Skill parameter etc. is also only an example in OK range, i.e. those skilled in the art can do suitable model by the explanation of this paper Interior selection is enclosed, and does not really want to be defined in hereafter exemplary specific data.

Cell used in the present invention is African green monkey kidney cell (Vero cell), is containing 10% fetal calf serum, 100U/L Penicillin, streptomysin DMEM culture medium in cultivate；

Zika virus used in the present invention (ZIKV, lower abbreviation virus) be Chinese pathogenic strain Z16006 (GenBank: 955589.1), it passes on and expands through Vero cell, -80 DEG C save for use；

Another: all tests relevant to live virus carry out in 2 grades of bio-safety of facility；

The chemical structural formula of Quercetin of the invention is as follows:

1, the cytotoxicity of Quercetin detects test:

Quercetin uses mtt assay to the toxicity detection of cell, specific as follows:

Vero cell is pressed 8 × 10³A/hole is inoculated in 96 orifice plates, at 37 DEG C, 5%CO₂Constant temperature cell incubator in Culture is about 80% to density, will be added in 96 orifice plates with the Quercetin of DMEM gradient dilution, every 200 μ L of hole continues to cultivate 96h discards cell culture supernatant, and the DMEM culture medium of 100 μ L MTT containing 0.5mg/mL is added in every hole, and 37 DEG C are continued to be incubated for 4h adds the conduct test group of Quercetin, is not added with Quercetin as a control group, uses multi-function microplate reader (Genios afterwards Pro, Tecan, US) detection 570nm at absorbance, the toxicity of Vero cell is referred to using the survival rate of cell as Quercetin Mark, according to formula: cell survival rate (%)=E/N × 100 calculates cell survival rate, wherein E is the absorbance of test group, and N is The absorbance of control group, as a result such as Fig. 1:

As shown in Figure 1: Quercetin in 0~12.5 μM of concentration range to Vero cell substantially without toxicity, Vero is thin The survival rate of born of the same parents maintains essentially in 100%, this illustrates that the biological safety of Quercetin is higher.

2, Quercetin inhibits zika virus infection cell and the mRNA expression of plaque effect and zika virus that generates Test:

In order to study the stage that Quercetin acts on zika virus life cycle, the present invention is ground with the following method Study carefully:

Vero cell is pressed 2 × 10⁵A/hole is inoculated in 12 orifice plates, at 37 DEG C, 5%CO₂Constant temperature cell incubator in Culture is about 80% to density, is divided into 4 groups of dosing methods:

A group: vero cells infection again after virus is incubated for Quercetin；

B group: virus infection Vero cell is added after Quercetin and Vero cell incubation；

C group: vero cells infection immediately after virus is mixed with Quercetin；

D group: Quercetin is added after virus infection Vero cell 1h；

Concrete operations are following (see Fig. 2):

A group: 100 μM of Quercetins first being mixed with the zika virus of 100TCID50 in equal volume, are incubated at room temperature 30min, It adds in Vero cell, at 37 DEG C, 5%CO₂After being incubated for 1h in constant temperature cell incubator, change into fresh without Quercetin DMEM culture medium；

B group: by 50 μM of Quercetins and Vero cell at 37 DEG C, 5%CO₂It is incubated for 1h in constant temperature cell incubator, discards Mongolian oak Pi Su, is added the zika virus of 100TCID50 at 37 DEG C, 5%CO₂It is incubated for 1h in constant temperature cell incubator, discards virus, is changed into The fresh DMEM culture medium without Quercetin；

C group: being added in Vero cell immediately after 100 μM of Quercetins are mixed in equal volume with the zika virus of 100TCID50, At 37 DEG C, 5%CO₂It is incubated for 1h in constant temperature cell incubator, discards virus, changes the fresh DMEM culture without Quercetin into Base；

D group: being inoculated in Vero cell for the zika virus of 100TCID50, at 37 DEG C, 5%CO₂In constant temperature cell incubator After being incubated for 1h, the fresh culture medium containing 50 μM of Quercetins is changed into；

Blank group is the cell culture that the DMSO that virus adds 0.31% with drug, only is not added；Virus control group is only to add Virus and 0.31% DMSO, the cell culture of drug is not added；

The final concentration of above-mentioned 4 groups of Quercetins is 50 μM.

Vero cell continues to cultivate 72h after the processing of above-mentioned 4 kinds of dosing methods, collects above-mentioned 4 groups of supernatant and does plaque Experiment detection progeny virus production, specifically: Vero cell is pressed 1.5 × 10⁵A/hole is inoculated in 12 orifice plates, 37 DEG C, 5%CO₂Constant temperature cell incubator in culture to density be about 80%, above-mentioned 4 groups of supernatants dilute 1000 times, are added In Vero cell, at 37 DEG C, 5%CO₂Constant temperature cell incubator be incubated for 1h, be added containing 1.2% methylcellulose, 2%FBS DMEM culture medium discards culture medium, 2% crystal violet (preparation of 4% paraformaldehyde) is added, places at room temperature after infecting 96h 30min is rinsed under flowing water, washes away unbonded Crystal Violet Dye, is finally taken pictures using enzyme-linked immunospot assay instrument；Together When, Vero cell total rna is extracted, is qRT-PCR to detect the mRNA expression of virus, as a result such as Fig. 3:

From Fig. 3 (A): the plaque effect that Quercetin generates the progeny virus of A group has apparent inhibiting effect, approaches 100% inhibitory effect, the plaque effect generated to tri- groups of B, C, D of progeny virus do not have inhibiting effect；

From Fig. 3 (B): Quercetin can significantly reduce the mRNA's of the intracellular zika virus of A group, part reduction C group Expression quantity, and the no significant difference of influence to the expression quantity of the mRNA of two groups of intracellular zika virus of B, D, with plaque reality The result tested is almost the same, after this illustrates that Quercetin may not be able to be adsorbed on cell and Quercetin cannot protect virus infection Cell, while also illustrating that Quercetin may act on zika virus enters cell stage (referred to as enter stage).

3, the stage of confirmation Quercetin effect zika virus:

Based on above-mentioned test, in order to further confirm that whether Quercetin acts on the stage that enters of zika virus, the present invention is also Following " administration timing of drug " test has been carried out, specifically:

Vero cell is pressed 2 × 10⁵A/hole is inoculated in 12 orifice plates, at 37 DEG C, 5%CO₂Constant temperature cell incubator in Culture to density is about 80%, and the zika virus of 100TCID50 is added in cell, at 37 DEG C, 5%CO₂Constant temperature cell training Support and be incubated in case, respectively at 0 (being added in cell simultaneously with virus), 1,2,3,4,5,8,12, for 24 hours, 50 μM of Quercetins are added, after It is continuous to be incubated for 1h, it discards supernatant, the DMEM culture medium of 2mL is added, continue to cultivate 72h, mention cell total rna, be qRT-PCR to detect The mRNA expression of zika virus, virus control group be only plus virus and 0.31% DMSO, the cell culture of drug is not added As a result object is shown in Fig. 4:

As shown in Figure 4: when virus infection 0h, Quercetin is most significant to the inhibitory effect of zika virus, and works as cell infection After zika virus, the inhibitory effect that Quercetin replicates zika virus is poor, this illustrates that Quercetin mainly acts on stockaded village again Block the stage that enters of virus, it is consistent with the test result of the relative expression quantity of the mRNA of above-mentioned zika virus.

4, the onset time after Quercetin is in conjunction with zika virus:

In order to determine Quercetin and the onset time after viral combined, the present invention is real using plaque assay and NS1 ELISA It tests and is studied, the specific method is as follows:

(1) Vero cell plaque assay: is pressed 1.5 × 10⁵A/hole is inoculated in 12 orifice plates, at 37 DEG C, 5%CO₂Perseverance Culture to density is about 80% in warm cell incubator, and 0.31% DMSO (control group) or 50 μM of Quercetin (experimental group) divides After not mixed with virus, it is added in Vero cell after being incubated for 0,1,5,10,30,60,120,240min respectively at room temperature, 37 DEG C, 5%CO₂Constant temperature cell incubator be incubated for 1h, be added containing 1.2% methylcellulose, 2%FBS DMEM culture medium, infection After 96h, implement plaque assay, be not added virus and drug, only plus 0.31% DMSO cell culture as blank control group, As a result such as Fig. 5 (A):

(2) NS1 ELISA is tested: Vero cell is pressed 8 × 10³A/hole is inoculated in 96 orifice plates, at 37 DEG C, 5%CO₂'s Culture to density is about 80% in constant temperature cell incubator, 0.31% DMSO (virus control group) or 50 μM of Quercetin (experiments Group) with virus mix be incubated for 0,1,5,10,30,60,120,240min respectively at room temperature after addition Vero cell in, disease is not added Poison and drug, only plus 0.31% DMSO cell culture as blank control group, at 37 DEG C, 5%CO₂Constant temperature cell training It supports case and is incubated for 1h, change fresh DMEM culture medium into, continue to cultivate 96h, discard cell culture supernatant, PBS washs 1 time, carefully Born of the same parents fix 20min with 4% paraformaldehyde at room temperature, discard paraformaldehyde, and the anhydrous methanol of 200 μ L frost is added in every hole, carefully Born of the same parents penetrating 5min under the conditions of 4 DEG C, is washed 1 time with cold PBS, and 2% skim milk that 150 μ L are added in every hole (contains 0.3% The PBS of Tween 20 is prepared), 1h is closed under the conditions of 37 DEG C, the PBS of 0.3%Tween 20 is washed 3 times, and 50 μ L are added in every hole NS1 primary antibody (1:2000), be incubated for 1h under the conditions of 37 DEG C；The PBS of 0.3%Tween 20 is washed 3 times, and 50 μ L bis- are added in every hole Anti- (1:2000), is incubated for 1h under the conditions of 37 DEG C；The PBS of 0.3%Tween 20 is washed 4 times, and the TMB solution of 50 μ L is added in every hole Develop the color 5min, is eventually adding the H of the 0.5mol/L of 50 μ L₂SO₄Solution terminates reaction, the use of microplate reader Detection wavelength is at 450nm Absorbance value, according to OD₄₅₀Size, judge that Quercetin inhibits the activity of cell entry, as a result such as Fig. 5 (B):

From Fig. 5 (A): Quercetin with virus mix after immediately (0min) infection cell do not have substantially compared with viral group Have and generate plaque effect, to the inhibiting rate almost 100% of virus；

From Fig. 5 (B): (0min) infection cell, energy 100% inhibit NS1 albumen immediately after Quercetin is mixed with virus Expression, this explanation consistent with the result of plaque assay work immediately after Quercetin and viral mix, and inhibiting rate is up to 100%.

5, the medium effective concentration (IC of the anti-zika virus infection of Quercetin In Vitro₅₀) detection:

Confirm that Quercetin acts on the cell entry stage by above-mentioned experiment, in order to detect the infection of effect of quercetin zika virus IC₅₀, the activity that the present invention infects zika virus using NS1 ELISA detection Quercetin, the specific method is as follows:

Vero cell is pressed 8 × 10³/ hole is inoculated in 96 orifice plates, at 37 DEG C, 5%CO₂Constant temperature cell incubator in train Supporting to density is about 80%；Vero is added after being incubated at room temperature 30min after mixing with virus in the Quercetin of various concentration gradient In cell, at 37 DEG C, 5%CO₂Constant temperature cell incubator be incubated for 1h, change fresh DMEM culture medium into, continue cultivate 96h, Cells and supernatant is discarded, NS1 ELISA detection is carried out, as a result such as Fig. 6, according to OD₄₅₀Data calculate quercitrin with Prism software Element inhibits the IC of virus infection₅₀Size.

As shown in Figure 6: Quercetin significantly inhibits zika virus, to viral inhibiting rate with Quercetin Concentration increase and increase, there are apparent dose-effect relationships, are calculated: medium effective concentration of the Quercetin to zika virus IC₅₀For IC₅₀=2.29 ± 0.55 μM.

6, influence of the Quercetin to zika virus NS2B-NS3 proteinase activity:

NS3 has important role to the processing maturation of pathogenicity, virus replication and protein.NS3 protease Region is the important nickase of the non-structural proteins such as NS3 synthesis, and it is multiple that NS3 protease need to be combined into NS2B-NS3 protease with NS2B The effect of object competence exertion enzymatic activity is closed, so NS2B-NS3 proteinase complex can be used as the important target of enzyme inhibitor class, base In this, the present invention studies influence of the Quercetin to zika virus NS2B-NS3 proteinase activity by the following method:

Construct NS2B-NS3 protease prokaryotic expression plasmid: NS2B-NS3 protease is thin in RosettaTM (DE3) competence It is expressed in born of the same parents, with the LB culture medium of ampicillin containing 100mg/L and 20mg/L chloramphenicol expanding bacterium under the conditions of 37 DEG C, (OD value is 0.6), then be added 0.5mM protein expression inducer IPTG under the conditions of 16 DEG C inducing expression 16h；

Expression and purification NS2B-NS3 protease: bacterium, ultrasound cracking bacterium are received to above-mentioned centrifugation, centrifugation retains supernatant, is added Nickel column combines, and is successively 125mM (1) with concentration, the protein lysis buffer of 250mM (2) and 500mM (3) imidazoles elutes, thoroughly Analysis removes imidazoles, with the protein solution after super filter tube concentration dialysis, finally crosses g75 molecular sieve using AKTA protein purification system, The peak with UV absorption is collected, destination protein purity is finally examined with SDS-PAGE；

Detection Quercetin is to the inhibiting effect of proteinase activity: above-mentioned NS2B-NS3 protease after purification is delayed with reacting Fliud flushing, series of concentrations Quercetin be pre-applied in the completely black plate in 96 holes and be incubated for 30min, Bz-Nle-Lys-Arg-Arg- is added AMC substrate, is incubated for 10min at room temperature, fluorescence intensity (RFU) in microplate reader, excitation wavelength 355nm, launch wavelength NS2B-NS3 protease is not added as a control group by 460nm, and Aprotinin inhibitor is added as positive controls, as a result sees Such as Fig. 7:

From Fig. 7 (A): it is examined through SDS-PAGE, obtains NS2B-NS3 protease purpose band in 25kDa or so, Purity reaches 80% or more；

From Fig. 7 (B): NS2B-G4TG4-NS3 recombinant protease (NS2B-NS3 protease) is added in experimental group, thus Strong fluorescence is generated, and buffer control group does not contain recombinant protease, substrate cannot be digested, and fluorescence signal is weaker, positive Control group, Aprotinin protease inhibition activity, the fluorescence signal of generation is also weaker, this illustrates what expression and purification obtained NS2B-G4TG4-NS3 recombinant protein enzymatic activity is preferable, meets inhibition of enzyme activity experiment；

From Fig. 7 (C): Quercetin can obviously inhibit NS2B-NS3 proteinase activity, to NS2B-NS3 protease activity Property inhibiting rate increase with the increase of concentration, there are apparent dose-effect relationship, Quercetin inhibits NS2B-NS3 protease IC₅₀1.012 ± 0.159 μM, 50 μM of Quercetin reaches 100% to the inhibiting rate of NS2B-NS3 protease.

In conjunction with the above result of study, Quercetin acts on zika virus and enters the stage, is a viral entry inhibitor, and NS2B-NS3 proteinase activity can obviously be inhibited, after this illustrates Quercetin probably by being combined with virus, interfere NS2B- NS3 proteinase activity hinders cell entry cell, and then influences the duplication of virus in the cell, therefore Quercetin can be used as one A potential anti-zika virus drug is developed.

Claims

1. Quercetin and its derivative are preparing the application in anti-zika virus drug.

2. application according to claim 1, it is characterised in that: the derivative of the Quercetin be its tautomer or its Pharmaceutically acceptable salt.

3. application according to claim 2, it is characterised in that: the Quercetin pharmaceutically acceptable salt is its alkali addition Salt is selected from sodium, potassium, calcium, magnesium, iron, ferrous iron, ammonium or zinc salt；Or acid-addition salts, it is selected from sulfate, acetate, hydrochloride, phosphoric acid Salt, oxalates, maleate, fumarate, malate, tartrate, citrate or benzoate.

4. application according to claim 1, it is characterised in that: the zika virus is Chinese pathogenic strain Z16006GenBank:955589.1。

5. application according to claim 1, it is characterised in that: further include that can pharmaceutically connect in the anti-zika virus drug Carrier, diluent, the excipient received.

6. application described in any one according to claim 1~5, it is characterised in that: the anti-zika virus drug is selected from mouth Take any one in agent, sustained release agent, tablet, pill, pulvis, granule, capsule, oral agents, suppository and injection.

7. a kind of drug of anti-zika virus, it is characterised in that: the drug includes Quercetin and/or its derivative.

8. drug according to claim 7, it is characterised in that: the derivative of the Quercetin be its tautomer or its Pharmaceutically acceptable salt.

9. drug according to claim 8, it is characterised in that: the Quercetin pharmaceutically acceptable salt is its alkali addition Salt is selected from sodium, potassium, calcium, magnesium, iron, ferrous iron, ammonium or zinc salt；Or acid-addition salts, it is selected from sulfate, acetate, hydrochloride, phosphoric acid Salt, oxalates, maleate, fumarate, malate, tartrate, citrate or benzoate.