CN105687226A - Preparation for inhibiting coronavirus infections - Google Patents

Preparation for inhibiting coronavirus infections Download PDF

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Publication number
CN105687226A
CN105687226A CN201610056160.7A CN201610056160A CN105687226A CN 105687226 A CN105687226 A CN 105687226A CN 201610056160 A CN201610056160 A CN 201610056160A CN 105687226 A CN105687226 A CN 105687226A
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preparation
coronavirus
cell
infection
egcg
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CN105687226B (en
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常国辉
刘京梅
杨益
黄留玉
罗丽晓
李瑾惠
罗彦军
孙走南
唐玥
苏文莉
张洁
刘璇
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Institute of Disease Control and Prevention of PLA
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/3533,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7024Esters of saccharides

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention discloses a preparation for inhibiting coronavirus infections. The preparation provided by the invention is applied to (1) or (2) as follows: (1) preparing of products for inhibiting the coronavirus infections; (2) inhibiting of coronavirus infections. The preparation is prepared mainly through mixing epigallocatechin gallate, tannin and astragalus polysaccharides, wherein the mass proportioning ratio of the epigallocatechin gallate to the tannin to the astragalus polysaccharides is (0.5 to 1.0): (0.5 to 1.0): (0.5 to 1.5). The cytotoxicity of the compounded preparation provided by the invention is not higher than that of a control sample, i.e., ribavirin which has already obtained security permission, so that the preparation is safer. The coronavirus infections can be effectively inhibited through using the preparation provided by the invention in a preventive manner in case of safe working concentration, so that the preparation has a commercial value in being further researched and developed into a coronavirus infection inhibitor.

Description

A kind of for suppressing the preparation of coronavirus infection
Technical field
The invention belongs to biological technical field, relate to a kind of preparation for suppressing coronavirus infection。
Background technology
Coronavirus is possible not only to cause breaking out of many animal epidemics, simultaneously, also being one of the main pathogen of mankind's common cold, childhood infection rate is higher, mainly upper respiratory tract infection, generally seldom involve lower respiratory tract, 2~5 days incubation periods, each age group all has morbidity, and child is common, with upper respiratory tract infection for feature, minority can cause diarrhoea, bronchitis, pneumonia and hydrothorax etc.。In recent years, especially meriting attention is the disease coronavirus respiratory system infection that usually causes that the mankind are serious, cause breaking out of the deadly infectious disease such as severe acute respiratory syndrome (SARS) and Middle East respiration syndrome (MERS), whole world public health system is caused great impact。
The reality and potentiality of facing mankind viral infectious threatens increasingly serious, the mankind tackle the sense multiplication of anti-governor pressure day of burst infectious disease, but more and more many practical situations show: when virus is undergone mutation or occurs that new virus causes that epidemic breaks out, specificity protective agents originally is difficult to performance should control effect, even has no curative effect, and the restriction in human cognitive level and scientific research cycle, the specificity of new generation prevention with significant curative effect and medicine is made again to be difficult to release at short notice, the calamity very easily causing diffusion and some areas rapidly of epidemic situation is broken out, not only local public health service is caused great destruction, also social politics and economy are caused huge impact simultaneously。Therefore, according to the Basic Biological Character of virus, for the general character target spot of virus infection host cell, researching and developing new virus infection inhibitor, the continuing challenge for tackling viral infectious epidemic situation is significant。
Plant compound is referred to as " the 7th class nutrient ", is widely present in conventional food, is class non-nutritive chemical substance that health has special role。Correlational study finds that Activities of Some Plants compound has the biological activity suppressing viral infection one of the focus being increasingly becoming antiviral study。Preliminary result of study shows: the biological effect of plant compound is mainly manifested in it to aspects such as the changes of the impact of cell membrane biological characteristic and local microenvironment balance, Activities of Some Plants compound is by being directly embedded into cell membrane lipid bilayer structure, change the normal functional flow of cell membrane and potential is poor, and then play certain biological effect。
Summary of the invention
It is an object of the invention to provide a kind of preparation suppressing coronavirus infection and application thereof。
Application provided by the present invention is specially the application in following (1) or (2) of a kind of preparation:
(1) preparation is for suppressing the product of coronavirus infection;
(2) it is used for suppressing coronavirus infection;
Described preparation is mainly mixed by epigallocatechin gallate (EGCG), tannic acid and astragalus polysaccharides;The quality proportioning of described epigallocatechin gallate (EGCG), described tannic acid and described astragalus polysaccharides is (0.5-1.0): (0.5-1.0): (0.5-1.5)。
In one embodiment of the invention, described for suppressing the preparation of viral infection specifically to be mixed by epigallocatechin gallate (EGCG) (EGCG), tannic acid and astragalus polysaccharides;The quality proportioning of described epigallocatechin gallate (EGCG) (EGCG), described tannic acid and described astragalus polysaccharides is 1:1:1.5。
In the present invention, described suppression coronavirus infection is following (a) or (b):
(a) prevention of infections by coronaviruses;
B () when simultaneously acting on host or host cell with coronavirus, it is suppressed that the infection to described host or described host cell of the described coronavirus。
More concrete, in an embodiment of the present invention, the inhibitory action of described coronavirus infection is embodied as by described preparation: using mammalian cell as coronavirus infection object, using described preparation while coronavirus infection cell or before coronavirus infection cell, the half-inhibition concentration of coronavirus is significantly reduced by described preparation compared with the positive control medicine suppressing viral infection。Described positive control medicine is specially ribavirin (Ribavirin)。
In the present invention, described mammalian cell (or the host cell described in (b)) is specially mouse fibroblast cell (17Cl-1 cell)。
In the present invention, described coronavirus is Mus hepatitis coronavirus。More concrete, described coronavirus is Mus hepatitis coronavirus A59 strain。
The present invention is with coronavirus (mousehepatitisvirus, MHV) for experimental subject, the method adopting observation of cell pathological changes, under different infectious conditions, analyze rule and the feature of epigallocatechin gallate (EGCG) (EGCG), tannic acid and astragalus polysaccharides these three plant compound monomer and mix preparation suppression viral infection thereof respectively。Result confirms: the cytotoxicity of three kind of plant compound monomers provided by the present invention and compound prescription mixture is all not higher than the control sample ribavirin obtaining security clearance, safer。Under the working concentration of safety, plant compound monomer and the plant extract compound prescription mixture such as preventive use EGCG and tannic acid, it is possible to the effective infection suppressing coronavirus。It is the commercial value of coronavirus infection inhibitor that preparation provided by the present invention has research and development further。
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method。
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain。
The quantitative experimental data related in following embodiment represents with mean ± standard deviation (± s), adopts SPSS17.0 statistical software to use the method for single factor test level variance analysis to carry out statistical disposition to comparing data between group。
Cell strain: mouse fibroblast cell (17Cl-1 cell), presented by University of Bristol of Britain professor StuartSiddell, it is recorded in " LinLei; SunYing; WuXiaoyan; SunZounan, YangYi, SuWenli; HuYi; ZhuQingyu, GuoDeyin, LiuJingmei, ChangGuohui.AttenuationofMouseHepatitisVirusbyDeletionof theLLRKxGxKGRegionofNsp1.PLoSONE, 20138 (4): e61166. " literary composition, the public can obtain from applicant, is only used for repeating the present invention and tests use。Cell is Secondary Culture according to a conventional method。
Virus: coronavirus (Mus hepatitis coronavirus A59 strain, it is called for short MHV-A59) it is recorded in " LinLei; SunYing; WuXiaoyan; SunZounan; YangYi; SuWenli, HuYi, ZhuQingyu, GuoDeyin, LiuJingmei, ChangGuohui.AttenuationofMouseHepatitisVirusbyDeletionof theLLRKxGxKGRegionofNsp1.PLoSONE, 20138 (4): e61166. " literary composition; the public can obtain from applicant, is only used for repeating the present invention and tests use。
Culture fluid: 1), Growth of Cells culture fluid: with DMEM culture medium (Glibo Products) for mother solution, it is separately added into 10% (volume fraction) hyclone (Sigma Products), 0.2mg/ml glutamine (Glibo Products), 100U/ml penicillin, streptomycin。2), cell maintenance culture solution, hyclone content is 2% (volume fraction), and all the other are with 1) all identical。3), virus multiplication culture fluid: identical with Growth of Cells culture fluid。
Epigallocatechin gallate (EGCG) (EGCG): Sigma Products (purity assay > 95%), production code member is E4143, CAS#989-51-5。
Tannic acid (Tannin): Sigma Products (purity assay > 99%), production code member is V900190;CAS#1401-55-4。
Astragalus polysaccharides (APS): Jin Sui bio tech ltd, Shanghai product (2-(Chloromethyl)-4-(4-nitrophenyl)-1,3-thiazole) (purity assay > 70%), production code member is JS11328;CAS#89250-26-0。
Positive control medicine: ribavirin (Ribavirin), Mei Lun biotech firm product。Because also there not being the standard positive control medicine suppressing viral infection by changing host cell membrane property at present。This research is using the most frequently used agents Ribavirin of current antiviral as comparison。Dissolve with PBS before experiment, make 2560 μ g/ml and filter, after degerming ,-20 DEG C of preservations。
Inverted phase contrast microscope: Japan's Olympus Products。
Cell culture incubator: U.S.'s Thermo Products。
Multifunctional enzyme instrument: MolecularDevices company of the U.S. multi-functional microplate reader of SpectraMaxM5 type。
1, cell recovery with go down to posterity
Taking out 17Cl-1 cell, 37 DEG C of water-baths in liquid nitrogen, after thawing, with the centrifugal 5min of 1000rpm, abandon supernatant, add suitable Growth of Cells culture fluid, piping and druming uniformly, makes cell density be about 2 × 10 repeatedly5Individual/ml, with 10ml/ bottle, in T25 Tissue Culture Flask, cellar culture in cell culture incubator。In Microscopic observation after 24h, the adherent situation of cell, after 48-72h, or when Growth of Cells to density is approximately 95%, abandon original fluid, add PBS, rinse twice, 0.5ml0.25% (0.25g/100ml) EDTA, incubation digestion in 37 DEG C of incubators, when cellular contraction becomes round, it is rapidly added Growth of Cells culture fluid, terminates digestion, repeatedly after piping and druming uniformly, it is centrifuged with 1000rpm, 5min, abandon supernatant, make suspension cell concentration be about 1 × 105Individual/ml, 10ml/ every bottle, moves into T25 Tissue Culture Flask, places cell culture incubator, carries out routine passage cultivation。
2, virus multiplication
Take the T25 Tissue Culture Flask that cell density is about 80%, abandon stock solution, with PBS, rinse twice, to remove serum residence, add virus dilution (diluent is PBS), inoculum concentration MOI=0.01, and 1ml culture medium, shake even, being placed in cell culture incubator and adsorb 1h, abandon supernatant, every bottle adds the corresponding virus multiplication culture fluid of 10ml。Observation of cell pathological changes, after 48-72h, or when cytopathy reaches more than 75%, receives poison, takes supernatant, multigelation 3 times, with 3000rpm, 4 DEG C, is centrifuged 5min, subpackage supernatant, and measures virus titer TCID50
3, virus titer TCID50Measure
Adopt cytopathogenic effect method (CPE) to measure, with single cell suspension, add in 96 hole microtest plates, every hole 200 μ l, make cell concentration reach 2 × 105Individual/ml, cultivates 48-72h in cell culture incubator, until when cell monolayer density is about 80%, taking out, abandoning culture medium, rinse twice with PBS, and utilizing virus multiplication culture fluid is 10 by 10 times of gradient dilutions of virus stock solution used-3~10-13, every Concentraton gradient 8 hole string, every hole 100 μ l, to set up blank two row, in cell culture incubator, adsorb 1h, abandon supernatant, rear addition maintains culture fluid 200 μ l。Every day Microscopic observation, the record experimental result when virus control group cytopathy is " ++++"。Calculate by Reed-Muench Liang Shi method, virus titer TCID50。Often organize strain, in triplicate。
lgTCID50Difference+lg (dilution factor higher than 50% pathological changes rate) between=(percent-50% higher than 50% pathological changes rate)/(percent-lower than the percent of 50% pathological changes rate higher than 50% pathological changes rate) × dilution factor logarithm。
Embodiment 1, plant extract compound prescription inhibitors of viral infection preparation
By epigallocatechin gallate (EGCG) (EGCG), tannic acid and astragalus polysaccharides according to the ratio mix homogeneously that quality proportioning is 1:1:1.5, obtain plant extract compound prescription preparation。
Dissolving with PBS before experiment, make 2560 μ g/ml (EGCG, tannic acid and astragalus polysaccharides total concentration in the solution) and filter, after degerming ,-20 DEG C save backup。
Embodiment 2, cell toxicity test
The present embodiment adopts dimethyl diaminophenazine chloride phagocytosis method to measure the compound prescription preparation of the embodiment 1 preparation cytotoxicity to mammalian cell。Concrete operations are as follows:
Compound prescription formulation soln (2560 μ g/ml) multiple proportions gradient dilution embodiment 1 prepared, obtains 20 μ g/ml, 40 μ g/ml, 80 μ g/ml, 160 μ g/ml, 320 μ g/ml, 640 μ g/ml, 1280 μ g/ml totally 6 concentration。Then it is added separately to different dilution diluents to cultivate 17Cl-1 cell and cell density is about in the 96 porocyte culture plates of 80%, every hole 100 μ l, each dilution factor all does 4 multiple holes, using normal cell (being namely not added with compound prescription preparation) as compareing, after effect 2h, abandon by test solution, add cell maintenance culture solution, every hole adds 200 μ l, is placed in cell culture incubator and cultivates, after 48h, every hole adds 0.1% (0.1g/100mL) dimethyl diaminophenazine chloride 25 μ l, after acting on 1.5h in 37 DEG C, by the liquid sucking-off in every hole, rinse 2 times with PBS。Every hole adds the cell pyrolysis liquid (acetic acid: ethanol: water=1:50:49 of 100 μ l, volume ratio), every hole adds 100 μ l, and set blank, utilize the multi-functional microplate reader of SpectraMaxM5 (MolecularDevices company of the U.S.), set absorbing light wavelength as 492nm, measure OD value, pass through one factor analysis of variance, relatively each concentration group and matched group (normal cell, namely compound prescription preparation it is not added with) significant difference between OD value, determine the maximal non-toxic concentration (i.e. the Cmax of no difference of science of statistics compared with matched group OD value) of compound prescription preparation prepared by embodiment 1。
Experiment arranges the monomer of epigallocatechin gallate (EGCG) (EGCG), tannic acid and astragalus polysaccharides these three compound simultaneously and compares as the monomer of compound prescription preparation;And viral infection resisting positive control drug ribavirin is set as positive control。
Result is as shown in table 1, it is seen that: the maximal non-toxic concentration of 17Cl-1 cell is 640 μ g/ml by compound prescription preparation, identical with the maximal non-toxic concentration of marketed drug ribavirin。Visible, the plant compound compound prescription preparation of embodiment 1 preparation has good safety。
The cell maximal non-toxic experimental result of table 1 plant compound monomer and compound prescription preparation
Embodiment 3, coronavirus infection inhibition test
The present embodiment adopts CPE method to measure the inhibition that coronavirus poison is infected by the compound prescription preparation of embodiment 1 preparation。It is Mus hepatitis coronavirus A59 strain for examination coronavirus。
Take and grow up to Monolayer growth of cells density and be about the cultivation of 80% and have 96 well culture plates of 17Cl-1 cell, outwell culture fluid, after rinsing cell 3 times with PBS, respectively to add the compound prescription preparation of embodiment 1 preparation under tri-kinds of conditions of A, B, C:
A. in viruses adsorption simultaneously: by isopyknic 2 × 100TCID50The virus liquid of coronavirus and 2 times of concentration the mixing of tested material solution after, in Tissue Culture Plate, add mixed liquor 100 μ l/ hole, in cell culture incubator, after viruses adsorption 1h, abandon it。Behind 3 times, PBS flushing cell face, add cell maintenance culture solution。
B., before viruses adsorption: every hole adds corresponding dilution tested material solution 100 μ l, abandons tested material solution, after PBS rinses cell face 3 times, addition titre is 100TCID50The virus liquid 100 μ l of coronavirus, in cell culture incubator, after viruses adsorption 1h, abandon it。Behind 3 times, PBS flushing cell face, add cell maintenance culture solution。
C. after viruses adsorption: 100TCID50The virus liquid 100 μ l/ hole of coronavirus, in cell culture incubator, after viruses adsorption 1h, abandon it。Add the every hole 100 μ l of corresponding dilution tested material solution, abandon tested material solution, after adsorbing 1h in cell culture incubator, abandon virus liquid。After PBS rinses 3 times, add cell maintenance culture solution。
Wherein, tested material is the compound prescription preparation of epigallocatechin gallate (EGCG) (EGCG), tannic acid and the monomer of astragalus polysaccharides these three compound, embodiment 1 preparation, or viral infection resisting positive control drug ribavirin。The concentration of tested material solution arranges following Concentraton gradient: 20 μ g/ml, 40 μ g/ml, 80 μ g/ml, 160 μ g/ml, 320 μ g/ml, 640 μ g/ml, 1280 μ g/ml。
Often (positive control is for only adding 100TCID for group Setup Experiments blank and positive controls50Virus, blank only adds cell maintenance culture solution), to cultivate in cell culture incubator, every day observes under inverted microscope, records experimental result when virus control group (i.e. positive controls) cytopathy is " ++++"。By Reed-Muench Liang Shi method calculation of half inhibitory concentration IC50
Result shows: when three kinds of mode administering modes, and EGCG, plant compound prescription preparation and ribavirin all may interfere with coronavirus infection 17Cl-1 cell。Wherein, when viruses adsorption while and when adding inhibitor before absorption, the effect of plant extract compound prescription preparation suppression viral infection is best, its virus half-inhibition concentration respectively 127 ± 17 μ g/ml and 107 ± 12 μ g/ml, and when adding inhibitor after viruses adsorption, the effect that comparison medicine ribavirin suppresses viral is better, and its virus half-inhibition concentration is 257 ± 31 μ g/ml。In monomer inhibitor, when EGCG adds before viruses adsorption, its infectious effect suppressing viral is best, and virus half-inhibition concentration is 172 ± 14 μ g/ml, when adding tannic acid and astragalus polysaccharides after viruses adsorption, all can not effectively suppress the infection of coronavirus。Concrete outcome is in Table 2。
The each inhibitor of table 2 is to coronavirus infection half-inhibition concentration (IC50)
Note: * represents and compares with ribavirin group, p < 0.05。
The experimental result of integrated embodiment 2 and 3, it is seen that: the cytotoxicity of compound prescription preparation provided by the present invention is not higher than the control sample ribavirin obtaining security clearance, safer。Under the working concentration of safety, preventive use plant extract compound prescription preparation provided by the present invention, it is possible to the effective infection suppressing coronavirus。It is the commercial value of coronavirus infection inhibitor that compound prescription preparation provided by the present invention has research and development further。
Comparative example, different ratio plant extract compound prescription preparation the inhibition of coronavirus infection is compared
Experimental technique is referring to embodiment 3, tested material is the compound prescription preparation of embodiment 1 preparation, comparison compound prescription preparation 1, comparison compound prescription preparation 2, comparison compound prescription preparation 3, or viral infection resisting positive control drug ribavirin, all the other operations are all with embodiment 3。Wherein, the formula of comparison compound prescription preparation 1, comparison compound prescription preparation 2 and comparison compound prescription preparation 3 is as follows:
Comparison compound prescription preparation 1: epigallocatechin gallate (EGCG), tannic acid and astragalus polysaccharides are mixed according to the ratio that quality proportioning is 0.5:1:0.5。
Comparison compound prescription preparation 2: epigallocatechin gallate (EGCG), tannic acid and astragalus polysaccharides are mixed according to the ratio that quality proportioning is 1:0.5:1。
Comparison compound prescription preparation 3: epigallocatechin gallate (EGCG), tannic acid and astragalus polysaccharides are mixed according to the ratio that quality proportioning is 1:1:1。
Result shows: comparison compound prescription preparation 1, comparison compound prescription preparation 2 and the comparison compound prescription preparation 3 half-inhibition concentration (IC to coronavirus infection50) under three kinds of conditions of A, B and C, all it is substantially less than compound prescription preparation (p < 0.05) prepared by embodiment 1。Concrete outcome is referring to table 3。
Table 3 different composite pharmaceutical formulation is to coronavirus infection half-inhibition concentration (IC50)
Note: * represents and compares with ribavirin group, p < 0.05。# represents and compares with embodiment 1 compound prescription preparation group, p < 0.05。

Claims (4)

1. the preparation application in following (1) or (2):
(1) preparation is for suppressing the product of coronavirus infection;
(2) it is used for suppressing coronavirus infection;
Described preparation is mainly mixed by epigallocatechin gallate (EGCG), tannic acid and astragalus polysaccharides;The quality proportioning of described epigallocatechin gallate (EGCG), described tannic acid and described astragalus polysaccharides is (0.5-1.0): (0.5-1.0): (0.5-1.5)。
2. application according to claim 1, it is characterised in that: described preparation is mixed by epigallocatechin gallate (EGCG), tannic acid and astragalus polysaccharides;The quality proportioning of described epigallocatechin gallate (EGCG), described tannic acid and described astragalus polysaccharides is 1:1:1.5。
3. application according to claim 1 and 2, it is characterised in that: described suppression coronavirus infection is following (a) or (b):
(a) prevention of infections by coronaviruses;
B () when simultaneously acting on host or host cell with coronavirus, it is suppressed that the infection to described host or described host cell of the described coronavirus。
4. according to described application arbitrary in claim 1-3, it is characterised in that: described coronavirus is Mus hepatitis coronavirus。
CN201610056160.7A 2016-01-27 2016-01-27 A kind of preparation for being used to suppress coronavirus infection Expired - Fee Related CN105687226B (en)

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