阅读说明：本技术 注射用丹参多酚酸盐在制备抗病毒的药物中的应用 (Application of salvianolate for injection in preparing antiviral drug ) 是由 刘叔文 杨婵 潘晓彦 许鑫锋 于 2020-05-18 设计创作，主要内容包括：本发明公开了一种注射用丹参多酚酸盐在制备抗病毒的药物中的应用。本发明首次提出注射用丹参多酚酸盐在制备抗病毒的药物中的应用,提供存在慢性基础疾病的新冠感染者新的治疗手段,该应用扩大了注射用丹参多酚酸盐的临床使用范围,且为病毒抑制,特别是在全球SARS-CoV-2流行的情况下,为治疗新冠病毒感染所致肺炎(COVID-19)提供新的药物。(The invention discloses an application of salvianolate for injection in preparing antiviral drugs. The invention firstly provides the application of the salvianolate for injection in preparing antiviral drugs, provides a new treatment means for new coronary infectors with chronic basic diseases, expands the clinical application range of the salvianolate for injection, and provides a new drug for virus inhibition, particularly for treating pneumonia (COVID-19) caused by new coronary virus infection under the condition of global SARS-CoV-2 epidemic.)

1. The application of salvianolic acid salt for injection in preparing antiviral medicines is provided.

2. The application of salvianolate for injection and other antiviral drugs in preparing antiviral drugs is provided.

3. The application of salvianolate for injection in preparing medicine for inhibiting virus from entering target cell is provided.

4. The application of the salvianolate for injection and other antiviral drugs in preparing drugs for inhibiting viruses from entering target cells.

5. The use of any one of claims 1 to 4, wherein the components of the salvianolate for injection comprise magnesium salvianolate, sodium rosmarinci, magnesium lithospermate, dipotassium lithospermate, potassium tanshinol, dipotassium salvianolate, dipotassium isosalvianolate, magnesium salvianolic acid G, and the content of the magnesium salvianolate is 80%;

the structural formula of the magnesium salvianolate is as follows:

the structural formula of the rosmarinic acid sodium is as follows:

the structural formula of the purple magnesium oxalate is as follows:

the structural formula of the dipotassium lithospermic acid is as follows:

the structural formula of the tanshinol potassium is as follows:

the structural formula of the red sage root dipotassium acetate is as follows:

the structural formula of the isosalvianic acid dipotassium is as follows:

the structural formula of the magnesium salvianolic acid G is as follows:

6. the use of any one of claims 1 to 4, wherein the virus comprises HIV-1 or coronavirus.

7. Use according to claim 6, wherein the coronavirus comprises SARS-CoV, SARS-CoV-2, MERS-CoV.

8. The use according to claim 7, wherein the coronavirus is SARS-CoV-2.

Technical Field

The invention belongs to the technical field of pharmacy, and particularly relates to application of salvianolate for injection in preparation of antiviral drugs.

Background

The novel coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 infection has become the global primary public health threat with the widest infection range and the greatest difficulty in prevention and control at present due to the extremely strong spreading rate and the ultra-long latency. SARS-CoV-2 belongs to the genus beta of coronavirus and is a linear single-stranded RNA virus (ssRNA). The genome sequence is about 30kb in length and contains 10 genes. The method comprises the following steps: ORF1ab, ORF1ab, S, ORF3a, E, M, ORF6, ORF7a, ORF8, N, ORF 10. The entry of SARS-CoV-2 into the host cell is mediated by the transmembrane Spike S glycoprotein (S), which exists as a trimer, each monomer consisting of one S1 and one S2 subunit, which is cleaved into S1 and S2 by the action of host cell proteases, wherein S1 binds to angiotensin converting enzyme 2(ACE2) of the host cell mediating the entry of the virus and the S2 subunit is responsible for the fusion of the virus and the cell membrane. Therefore, S protein inhibitors can prevent viruses from entering host cells, and S1 and S2 subunits can be used as targets for screening antiviral drugs. Currently, there is no specific drug against SARS-CoV-2 and no approved drug on the market.

Salvia miltiorrhiza (Salvia miltiorrhizae Bunge) is a traditional Chinese medicine with the function of promoting blood circulation and removing blood stasis which is used in clinic for a long time, a water-soluble extraction component of Salvia miltiorrhiza polyphenol acid salt for injection obtains a new medicine certificate of raw materials and preparations in 5 months in 2005, is a new medicine of Chinese medicine class II developed by Shanghai medicine research institute of Chinese academy of sciences through 13 years research, and is produced by Shanghai green valley pharmacy Limited company. The product is put into the market in 2006, has the functions of mainly treating stable angina pectoris of coronary heart disease, is classified into I and II, the symptoms of angina pectoris are mild and moderate, and the syndrome differentiation of traditional Chinese medicine is heart blood stasis syndrome. Is also suitable for patients with acute myocardial infarction Percutaneous Coronary Intervention (PCI), and can reduce non-refluence, improve myocardial microcirculation, etc.

The 'Salvia miltiorrhiza polyphenol acid salt for injection' is polyphenol acid salt with magnesium Salvia miltiorrhiza acetate as the main active component, wherein the content of magnesium Salvia miltiorrhiza acetate is as high as 80%, the pharmacological activity of the salt is superior to that of acid, magnesium ions are the important material basis of the pharmacodynamic action of the salt, and the rest 20% of the components are magnesium Violate, sodium Rosmarinate, dipotassium Salvia miltiorrhiza acetate, dipotassium IsoSalvia miltiorrhiza acetate, dipotassium Violet oxalate, potassium Salvianic acid and magnesium Salvianolic acid G. In the production process, magnesium salvianolate is used as a quality control standard, and the quality of medicinal materials, raw material medicaments and preparations is comprehensively controlled by using a fingerprint spectrum technology. Meanwhile, the preparation adopts a freeze-dried powder technology, avoids the instability of the compound, and ensures that the quality standard of the product is obviously higher than that of the similar traditional salvia miltiorrhiza preparation.

Chinese patent No.: cn.102058599 has reported a preparation method of salvianolate for injection: extracting Saviae Miltiorrhizae radix with 50-95% (V/V) ethanol for three times, each for 2 hr, filtering the extractive solution, concentrating under reflux to obtain concentrated solution with relative density of 1.00-1.20 at 50-60 deg.C, diluting with water to obtain concentrated solution with relative density of 0.92-1.12 at room temperature, adsorbing with polydivinylbenzene type macroporous adsorbent resin, washing with water to remove sugar, protein and inorganic salts, eluting Saviae Miltiorrhizae radix polyphenol acid salt compound other than Saviae Miltiorrhizae radix acetate with ethanol of below 10% (V/V), eluting Saviae Miltiorrhizae radix acetate with 15-25% ethanol, collecting and concentrating eluate, precipitating with 90% ethanol, filtering to obtain filtrate, and vacuum drying to obtain the final product.

The salvianolate for injection can improve microcirculation through calcium channel block, inhibit thromboxane A2(TXA2) synthetase and inhibit platelet aggregation, and is widely applied to the treatment of clinical cardiovascular and cerebrovascular system diseases. In addition, based on the fact that salvianolate contains a large number of phenolic hydroxyl groups, has the effects of scavenging free radicals and strong antioxidation, and has been verified to have anti-inflammatory and liver-protecting effects. But has not been studied in the field of antiviral so far.

Disclosure of Invention

The invention aims to provide a novel antiviral drug.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention provides an application of salvianolate for injection in preparing antiviral drugs.

The invention also provides application of the salvianolate for injection and other antiviral drugs in preparation of antiviral drugs.

The invention also provides application of the salvianolate for injection in preparing a medicament for inhibiting viruses from entering target cells.

The invention further provides an application of the salvianolate for injection and other antiviral drugs in preparation of drugs for inhibiting viruses from entering target cells.

Further, the salvianolate for injection comprises magnesium salvianolate, sodium rosmarinci acid, magnesium lithospermate, dipotassium lithospermate, potassium tanshinol, dipotassium salvianolate, dipotassium isosalvianolate and magnesium salvianolic acid G, wherein the content of the magnesium salvianolate is 80%;

the structural formula of the magnesium salvianolate is as follows:

the structural formula of the rosmarinic acid sodium is as follows:

the structural formula of the purple magnesium oxalate is as follows:

the structural formula of the dipotassium lithospermic acid is as follows:

the structural formula of the tanshinol potassium is as follows:

the structural formula of the red sage root dipotassium acetate is as follows:

the structural formula of the isosalvianic acid dipotassium is as follows:

the structural formula of the magnesium salvianolic acid G is as follows:

further, the virus includes HIV-1 or coronavirus.

Further, the coronavirus includes SARS-CoV, SARS-CoV-2, MERS-CoV.

Further, the coronavirus is SARS-CoV-2.

The invention has the following beneficial effects:

the invention firstly provides the application of the salvianolate for injection in preparing antiviral drugs, provides a new treatment means for new coronary infectors with chronic basic diseases, expands the application range of the salvianolate for injection, and provides a new drug for virus inhibition, particularly for treating pneumonia (COVID-19) caused by new coronary virus infection under the condition of global SARS-CoV-2 epidemic.

The salvianolate for injection is used as an antiviral drug, and experiments show that: injecting half effective concentration EC of salvianolate for resisting SARS-CoV-2 activity on Vero-E6 infection model of in vitro cultured cell₅₀36.07 μ g/ml; injection salvianolate for inhibiting SARS-CoV-2 entry stage, and inhibiting SARS-CoV-2S pseudovirus activity, IC₅₀19.81. mu.g/ml. In addition, no significant cytotoxicity was observed in the effective concentration range. Therefore, the invention can be used for preparing anti-SARS-CoV-2 medicine, and has larger clinical application value.

Drawings

FIG. 1 is a graph showing the inhibition rate of Salvianolic acid salt for injection (ZDDY) against SARS-CoV-2 at different concentrations in example 1 of the present invention, wherein the abscissa represents the concentration of Salvianolic acid salt for injection (ZDDY), and the ordinate represents the inhibition rate of Salvianolic acid salt for injection (ZDDY) against SARS-CoV-2 with respect to a solvent group, and the half effective concentration EC of Salvianolic acid salt for injection (ZDDY) against SARS-CoV-2 is calculated from the inhibition rates₅₀The value is obtained.

FIG. 2 is a graph showing the inhibition rate of salvianolic acid salt (ZDDY) for injection against entering into target cells of SARS-CoV-2S protein pseudovirus at different concentrations in example 2, wherein the abscissa represents the concentration of salvianolic acid salt (ZDDY) for injection, and the ordinate represents the inhibition rate of salvianolic acid salt (ZDDY) for injection against entering of SARS-CoV-2S protein pseudovirus using solvent group as control, and the half-inhibitory concentration IC50 value of salvianolic acid salt (ZDDY) for injection against entering of SARS-CoV-2S protein pseudovirus is determined.

FIG. 3 is a graph showing the survival rate of Salvianolic acid salt for injection (ZDDY) versus Vero-E6 cells as target cells in example 3 of the present invention, wherein the abscissa represents the concentration of Salvianolic acid salt for injection (ZDDY) and the ordinate represents the percentage of cells surviving after administering different concentrations of Salvianolic acid salt for injection (ZDDY) to Vero-E6 cells with the control solvent group.

FIG. 4 is a graph showing the survival rate of 293T/ACE2 cells as target cells after injection of salvianolate (ZDDY) in example 3, wherein the abscissa represents the concentration of salvianolate (ZDDY) and the ordinate represents the percentage of cells surviving 293T/ACE2 cells after administration of different concentrations of salvianolate (ZDDY) in the solvent group as a control.

Detailed Description

For a better understanding of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawings, and the scope of the invention is not limited to the following examples.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The following examples mainly establish SARS-CoV-2 live virus and SARS-CoV-2S pseudovirus in vitro cell infection model to evaluate the anti-SARS-CoV-2 activity of salvianolate for injection, and confirm that salvianolate for injection has the ability of anti-SARS-CoV-2 infection, and simultaneously has the process of inhibiting SARS-CoV-2 from entering target cells, and provides an application of salvianolate for injection in preparing anti-novel coronavirus medicine.

Vero-E6 and 293T cells adopted by the invention are purchased from American ATCC, and 293T cells stably over-expressing human SARS-CoV-2 receptor protein ACE2 are constructed and stored by the unit.

The cell growth culture solution adopted in the embodiment of the invention comprises the following components: DMEM basal medium, wherein fetal bovine serum with a total volume of 10% and ampicillin/streptomycin with a total volume of 1% are added, and the culture solution is stored at 4 ℃ and preheated in a water bath at 37 ℃ before use.

The salvianolate for injection adopted in the embodiment of the invention is produced by Shanghai green valley pharmacy Co., Ltd, and 100mg (80 mg containing salvianolic acid B magnesium) is bottled. The execution standard is the national food and drug administration standard YBZ09012005-2010Z and the national drug standard Z20050248.

SARS-CoV-2 used in the examples of the present invention was isolated from infected persons who were studied for the Wuhan virus and amplified for storage.

Pseudovirus packaging plasmids and sources thereof in the examples of the invention: the pseudovirus packaging skeleton plasmid pNL4-3.Luc. R-E-is certified and preserved by southern medical university, and the disclosed optimized full-length SARS-CoV-2S protein core plasmid pcDNA3.1-SARS-CoV-2-Sipke is a gift offered by professor Luway of Shanghai double-den university.

The luciferase assay kit adopted in the embodiment of the invention is purchased from Promega corporation of America and comprises a luciferase substrate and a cell lysate.

The Takara MiniBEST Viral RNA/DNA Extraction Kit, Takara PrimeScript RT reagent Kit with gDNA Eraser, Takara TBPremix Ex Taq^TMIITliRNaseH Plus was purchased from Takara.

Pharmacological experiment part

EXAMPLE 1 in vitro Activity assay of Salvianolic acid salt for injection against SARS-CoV-2

1. The method comprises the following steps:

1) Vero-E6 cells in logarithmic growth phase were seeded in 48-well plates at 3X 10^5 cells/well at 37 ℃ with 5% CO₂The culture was carried out overnight.

2) Pre-hatching with medicaments: the drug was diluted in DMEM medium containing 2% by volume fetal bovine serum. Initial concentration of salvianolate for injection (ZDDY) was set at 300. mu.g/ml (DMSO as solvent), three-fold dilutions of drug were made, with 3 multiple wells per concentration of drug, for a total of 7 drug gradients (300, 100, 33.33, 11.11, 3.70, 1.23, 0.41. mu.g/ml). Solvent dimethyl sulfoxide (DMSO) was set as a control, and the control group was diluted with DMEM medium containing 2% fetal bovine serum in total volume and administered with the same volume of dimethyl sulfoxide as the drug. After removing cell supernatant 1), 100. mu.l of diluted drug was added to each well of the experimental group in 48-well plate, 100. mu.l of diluted DMSO was added to the control group, and incubation was performed at 37 ℃ for 1 h. .

3) Viral infection: mu.l of SARS-CoV-2 virus dilution (MOI 0.05) was added to each well of the 48-well plate, and the cells were incubated at 37 ℃ for 1 h.

4) Liquid changing: the infected supernatant was removed well and the cells were washed once with 200. mu.l PBS. 200. mu.l of the medium containing the drug at the corresponding concentration was added to each well, and 150. mu.l of the cell culture supernatant was collected after further culturing at 37 ℃ for 24 hours. And (3) determining the copy number of the virus in the supernatant by adopting qRT-PCR (quantitative reverse transcription-polymerase chain reaction), and evaluating the capability of the medicine for resisting SARS-CoV-2 live virus.

5) The specific operation method for Viral RNA extraction was described in reference to Takara MiniBEST Viral RNA/DNAextraction Kit (Code No. 9766):

a) splitting the virus: mu.l of cell culture supernatant was supplemented to 200. mu.l with 50. mu.l of PBS (pH 7.4). Then 200. mu.l of Buffer VGB, 20. mu.l of protease K and 1.0. mu.l of Carrier RNA were added, mixed well and incubated in a 56 ℃ water bath for 10 minutes for sufficient lysis. Add 200. mu.l absolute ethanol to the lysate, suck well and mix well.

b) The Spin Column was mounted on a Collection Tube, the solution was transferred to the Spin Column, centrifuged at 12,000rpm for 2 minutes, and the filtrate was discarded.

c) Mu.l of Buffer RWA was added to the Spin Column, centrifuged at 12,000rpm for 1 minute, and the filtrate was discarded.

d) Mu.l of Buffer RWB was added to the Spin Column, centrifuged at 12,000rpm for 1 minute, and the filtrate was discarded. (the Buffer RWB had added a specified volume of 100% ethanol). Buffer RWB was added around the Spin Column wall to help completely flush out salt adhering to the wall.

e) And d, repeating the operation step.

f) Spin Column was mounted on the Collection Tube and centrifuged at 12,000rpm for 2 minutes.

g) The Spin Column was mounted on a new 1.5ml RNase free collection tube, and 30. mu.l of RNase free dH was added to the center of the Spin Column membrane₂And O, standing for 5 minutes at room temperature. The RNA was eluted by centrifugation at 12,000rpm for 2 minutes.

6) Specific procedures for reverse transcription of viral RNA (see Takara PrimeScriptTM RT reagent Kit with gDNA Eraser, Code No. RR047A):

a) removing genome DNA reaction: the following components were mixed on ice to prepare a reaction mixture

Reagent	Volume (μ l)
		5*gDNA Eraser Buffer	2.0
gDNA Eraser	1.0
		Total RNA	3.0
RNase Free dH2O	4.0
		Total volume	10.0

The sample was left to react at 42 ℃ for 2 min.

b) Reverse transcription reaction system: on ice configuration

Reagent	Volume (μ l)
		Reaction solution of step 1	10.0
PrimeScript RT Enzyme Mix I	1.0
		RT Primer Mix	1.0
5×PrimeScript Buffer 2(for Real Time)	4.0
		RNase Free dH2O	Is supplemented to 20.0

The samples were incubated at 37 ℃ for 15min and then heated at 85 ℃ for 5 sec.

7) Virus copy number detection using qPCR: reference is made to Takara TBPremix Ex Taq^TMII (TliRNaseH Plus, Code No. RR820A) (Standard Curve method: RBD plasmid of known copy number was used as a standard, and specific primers were targeted to RBD). The reaction solution was prepared on ice as follows:

the primer sequences are as follows:

RBD upstream Primer (Forward Primer): CAATGGTTTAACAGGCACAGG (SEQ ID NO: 1)

RBD downstream Primer (Reverse Primer): CTCAAGTGTCTGTGGATCACG (SEQ ID NO: 1)

According to the standard program of two-step PCR amplification, the detection is completed on an ABI7500 quantitative PCR instrument:

stage 1: pre-denaturation, Reps: 1 cycle, 95 ℃,30 s;

stage 2: PCR reaction, Reps: 40 cycles, 95 ℃,5 s;

annealing: 60 ℃ for 30-34 seconds.

2. As a result: as shown in fig. 1;

the copy number of each sample was calculated from the standard curve. The drug-treated group inhibition rate was calculated with DMSO group copy number as a reference. Fitting a drug inhibition rate curve by using prism8.0 software according to the inhibition rates of drug treatment groups with different concentrations, and calculating the half effective concentration EC of the salvianolate (ZDDY) for injection for resisting SARS-CoV-2 activity₅₀36.07. mu.g/ml.

Example 2 detection of inhibitory Activity of Salvianolic acid salt for injection against entry of SARS-CoV-2-S protein pseudovirus

1. The method comprises the following steps:

1) SARS-CoV-2S protein (pNL4-3.Luc. R-E-pcDNA3.1-SARS-CoV-2-Sipke) pseudovirus packaging:

HEK-293T cells in logarithmic growth phase 4 x 10^ 5/ml, 2ml per well were seeded in 6-well plates. 37 ℃ and 5% CO₂The cells were cultured in a cell incubator for 24 hours. Fresh culture medium is replaced 1 hour before transfection, 100 mul of blank DMEM culture medium is respectively adopted to prepare plasmid diluent and transfection reagent (PolyJet) diluent, and the preparation proportion of each well is as follows (plasmid DNA needs to be extracted by an extraction kit for removing endotoxin):

pNL4-3.Luc.R-E- 1000ng

pcDNA3.1-SARS-CoV-2-Sipke 500ng

PolyJet 6μl

the preparation method comprises the following steps: the pNL4-3.Luc. R-E-plasmid and pcDNA3.1-SARS-CoV-2-Sipke plasmid were added into 100. mu.l of blank DMEM medium at the same time and mixed, and Polyjet was diluted with 100. mu.l of blank DMEM medium and mixed. Adding the PolyJet diluent into the plasmid diluent, uniformly mixing, incubating for 15 minutes at room temperature, uniformly adding into HEK-293T cells, culturing for 48 hours at 37 ℃, collecting supernatant virus liquid, centrifuging for 10 minutes at 4000rpm, and filtering by using a 0.45-micrometer sterile filter head to obtain the SARS-CoV-2 pseudovirus.

2) Pseudovirus inhibition experiments:

drug and pseudovirus effects: taking 293T cells (293T/ACE2) which overexpress SARS-CoV-2 receptor ACE2 in logarithmic growth phase, and performing expression at 1 × 10⁴One/well was plated evenly in 96-well cell plates. Culturing in 37 ℃ cell culture boxAnd culturing for 24 hours.

The initial concentration of salvianolic acid salt for injection is set to be 25 mug/ml, 7 concentration gradients (25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.390625 mug/ml) are diluted by 2 times of DMEM medium containing 2 percent of fetal bovine serum in total volume before administration, 60 mug of each well and 3 multiple wells of each concentration, and a DMSO solvent control group is set. 60 mul of pseudovirus is added into the diluted medicine, mixed evenly and acted for 30 minutes at room temperature, 100 mul/hole is added into ACE2/293T cells, and the cells are cultured for 48 hours at 37 ℃.

And (3) detection: the medium was removed and the cells were washed once with 200. mu.l/well sterile PBS (pH7.4), 40. mu.l of 1X cell lysate was added to each well and lysed with shaking at room temperature for 15 minutes. Transferring 30 mul/hole cracking supernatant to a 96-hole white enzyme label plate, adding an isovolumetric diluted luciferase substrate according to the specification of a single luciferase detection kit, immediately detecting the fluorescence value by an enzyme label, and judging the activity of the salvianolate for injection for inhibiting virus adsorption according to the fluorescence value. Calculating the inhibition rate according to the corresponding relation between the fluorescence value and the drug concentration, drawing a curve and calculating the half inhibition concentration IC of the salvianolate for injection₅₀。

2. As a result: as shown in fig. 2;

and (5) calculating the inhibition rate of the drug treatment group according to the fluorescence value by taking the DMSO solvent group as a control. Fitting a drug inhibition rate curve by using prism8.0 software according to the inhibition rates of drug treatment groups with different concentrations, and calculating the half effective inhibition concentration IC of salvianolate (ZDDY) for injection for inhibiting SARS-CoV-2S protein pseudovirus from entering target cells₅₀19.81. mu.g/ml.

Example 3 cytotoxicity assay of Salvianolic acid salt for injection

1. The method comprises the following steps:

1) cell inoculation:

Vero-E6, 293T/ACE2 cells in logarithmic growth phase, adjusted cell density to 1x 10^ 4/well, seeded at 100. mu.L/well in 96-well plates, cultured overnight in 37 ℃ cell culture box.

2) Designing the concentration of the medicine:

before administration, 7 concentration gradients were diluted 2-fold in DMEM medium containing 2% total volume of fetal bovine serum.

Vero-E6 cells: initial concentrations were set at 300. mu.g/ml (300, 100, 33.33, 11.11, 3.70, 1.23, 0.41. mu.g/ml) and 100. mu.L of diluted drug per well was added to 1, respectively), to Vero-E6 cells in 96-well plates in a final volume of 200. mu.L per well. 3 multiple wells were set for each drug concentration. The DMSO solvent treated group served as blank control.

293T-ACE2 cells: initial concentrations were set at 250. mu.g/ml (250, 125, 62.5, 31.25, 15.625, 7.813, 3.906. mu.g/ml) and 100. mu.L of diluted drug per well was added to 293T-ACE2 cells in 96-well plates at a final volume of 200. mu.L per well. 3 multiple wells were set for each drug concentration. The DMSO solvent treated group served as blank control.

3) Detecting the absorbance:

after 48h of incubation in the incubator, 10. mu.L of CCK-8 working solution was added to each well and the incubator was incubated for 3 hours. And (5) measuring the absorbance at 450nm by using a microplate reader.

4) Based on the measured OD values, the survival rates of Vero-E6 and 293T-ACE2 cells at the respective concentrations of the drugs were calculated, respectively, as compared with the control group.

2. As a result: as shown in fig. 3 and 4;

salvianolic acid salt (ZDDY) for injection was administered to Vero-E6 cells at 300. mu.g/ml and in an effective concentration range (FIG. 3). Salvianolic acid salt (ZDDY) for injection has no obvious toxic effect on 293T/ACE2 cells (figure 4) in 250 μ g/ml and effective concentration range.

The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.

SEQUENCE LISTING

<110> southern medical university

Wuhan Institute of Virology, Chinese Academy of Sciences

<120> application of salvianolate for injection in preparing antiviral drug

<130> CP120010293C

<160> 2

<170> PatentIn version 3.3

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caatggttta acaggcacag g 21

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ctcaagtgtc tgtggatcac g 21