阅读说明：本技术 丹参素或其药物可接受盐在制备抗病毒的药物中的应用 (Application of tanshinol or its pharmaceutically acceptable salt in preparing antiviral medicine ) 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：本发明公开了一种丹参素或其药物可接受盐在制备抗病毒的药物中的应用。本发明首次提出丹参素或其药物可接受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,该应用扩大了丹参素的使用范围,且为病毒抑制,特别是在全球新冠流行的情况下,为抑制SARS-CoV-2提供新的药物。(The invention discloses an application of danshensu or a pharmaceutically acceptable salt thereof in preparing antiviral drugs. The invention firstly provides the application of the tanshinol or the pharmaceutically acceptable salt thereof in preparing antiviral medicaments, provides a new treatment means for new crown infectors with chronic basic diseases, expands the application range of the tanshinol, and provides a new medicament for inhibiting viruses, particularly SARS-CoV-2 under the condition of global new crown epidemic.)

1. Application of tanshinol or its pharmaceutically acceptable salt in preparing antiviral medicine is described, wherein the formula of tanshinol is C₉H₁₀O₅Molecular weight of 198.17, and structural formula

2. Application of tanshinol or its pharmaceutically acceptable salt in preparing medicine for inhibiting virus from entering target cell, wherein the formula of tanshinol is C₉H₁₀O₅Molecular weight of 198.17, and structural formula

3. Use according to claim 1 or 2, wherein the virus is a coronavirus.

4. The use according to claim 3, wherein the virus is SARS-CoV-2.

5. An antiviral pharmaceutical composition comprising tanshinol or a pharmaceutically acceptable salt thereof as an active ingredient, wherein said tanshinol has the molecular formula of C₉H₁₀O₅Molecular weight of 198.17, and structural formula

6. A pharmaceutical composition for inhibiting the entry of a virus into a target cell, comprising as an active ingredient a danshensu of formula C or a pharmaceutically acceptable salt thereof₉H₁₀O₅Molecular weight of 198.17, and structural formula

7. The pharmaceutical composition of claim 5 or 6, wherein the virus is a coronavirus.

8. The pharmaceutical composition of claim 7, wherein the virus is SARS-CoV-2.

9. The pharmaceutical composition according to claim 5 or 6, wherein the pharmaceutical composition is an injectable formulation or an oral formulation.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is an injection, a tablet, a capsule, a pill or a drop pill.

Technical Field

The invention belongs to the technical field of pharmacy, and particularly relates to application of danshensu or pharmaceutically acceptable salt thereof in preparing antiviral drugs.

Background

SARS-CoV-2 is a single-stranded RNA positive-strand enveloped beta coronavirus, the 7 th known coronavirus capable of infecting human is known at present, and the genome total length is about 26-32 kb. The entry of SARS-CoV-2 into the host cell is mediated by the transmembrane Spike S glycoprotein (S), which is the key to viral infection, and therefore the development of the structure of this S protein is a primary step in drug development. The S glycoprotein comprises two functional subunits, S1 and S2, of which S1 is responsible for binding to host cell receptors and S2 is responsible for viral membrane and cell membrane fusion. At present, no specific medicine aiming at SARS-CoV-2 exists, no medicine is approved to be on the market, and no SARS-CoV-2 entry inhibitor targeting S protein from natural products is reported.

Salvia miltiorrhiza (Salvia miliiorrhiza Bunge), a perennial upright herb of the genus Salvia of the family Labiatae, can be used as a drug for both its root and rhizome, and is classified into two categories according to the characteristics and physicochemical properties of the isolated compounds: fat-soluble tanshinone compound and water-soluble phenolic acid compound. Salvianic acid A is one of main effective components in water soluble components of Saviae Miltiorrhizae radix, is a phenolic aromatic acid compound separated from radix Salviae Miltiorrhizae in 1980, named as Laelia acid A, and the pharmacological action research of Salvianic acid is mainly focused on discussing cardiovascular activity characteristics and action mechanism. It has effects in dilating coronary artery, inhibiting platelet aggregation, scavenging oxygen free radicals, and improving memory disorder. However, there is no application in the prevention or treatment of the novel coronavirus.

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 danshensu or pharmaceutically acceptable salt thereof in preparing antiviral drugs, wherein the molecular formula of danshensu is C₉H₁₀O₅Molecular weight of 198.17, and structural formula

In another aspect, the invention provides an application of danshensu or a pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting viruses from entering target cells, wherein the molecular formula of danshensu is C₉H₁₀O₅Molecule(s)In an amount of 198.17, the formula

Further, the virus is a coronavirus.

Further, the virus is SARS-CoV-2.

In still another aspect, the present invention provides an antiviral pharmaceutical composition comprising danshensu of the formula C or a pharmaceutically acceptable salt thereof as an active ingredient₉H₁₀O₅Molecular weight of 198.17, and structural formula

In still another aspect, the present invention provides a pharmaceutical composition for inhibiting viral entry into a target cell, comprising danshensu of formula C or a pharmaceutically acceptable salt thereof as an active substance₉H₁₀O₅Molecular weight of 198.17, and structural formula

Further, the virus is a coronavirus.

Further, the virus is SARS-CoV-2.

Further, the pharmaceutical composition is an injection preparation or an oral preparation.

Further, the pharmaceutical composition is a prescription injection, a prescription tablet, a prescription capsule, a prescription pill or a prescription drop pill.

The invention has the following beneficial effects:

the invention firstly provides the application of the tanshinol or the pharmaceutically acceptable salt thereof in preparing antiviral medicaments, provides a new treatment means for new crown infectors with chronic basic diseases, expands the application range of the tanshinol, and provides a new medicament for inhibiting viruses, particularly SARS-CoV-2 under the condition of global new crown epidemic.

The experiment shows that the danshensu can effectively inhibit SARS-CoV-2 on in vitro cultured cell Vero-E6 and has half effective concentration EC₅₀1.07 μ M; salvianic acid A is used for inhibiting SARS-CoV-2 entry stage, inhibiting SARS-CoV-2S protein pseudovirus infection 293T/ACE2 cell, and inhibiting half inhibitory concentration IC₅₀2.70. mu.M; the danshensu has no obvious cytotoxicity in the effective concentration range. Therefore, can be used for preparing anti-SARS-CoV-2 medicine.

Drawings

FIG. 1 is a graph showing the inhibition rate of tanshinol against SARS-CoV-2 at different concentrations in example 1 of the present invention, in which the abscissa represents the tanshinol concentration and the ordinate represents the inhibition rate of tanshinol against SARS-CoV-2 using a solvent group as a control, and the half effective concentration EC of tanshinol against SARS-CoV-2 is calculated from the inhibition rates₅₀The value is obtained.

FIG. 2 is a graph of the inhibition rate of tanshinol at different concentrations for inhibiting SARS-CoV-2S protein pseudovirus from entering target cells in example 2 of the present invention, in which the abscissa represents the tanshinol concentration and the ordinate represents the inhibition rate of tanshinol for inhibiting SARS-CoV-2S protein pseudovirus from entering, using solvent group as control, and the half inhibition concentration IC50 value of tanshinol for inhibiting SARS-CoV-2S protein pseudovirus from entering is determined.

FIG. 3 is a graph showing the survival rate of the cells Vero-E6 cells, which are target cells, in example 3 of the present invention, wherein the abscissa represents the concentration of tanshinol, and the ordinate represents the percentage of survival of Vero-E6 cells after administration of different concentrations of tanshinol, using the solvent group as a control.

FIG. 4 is a graph showing the survival rate of 293T/ACE2 cells as target cells in example 3, wherein the abscissa represents the concentration of tanshinol and the ordinate represents the percentage of cell survival of 293T/ACE2 cells after administration of different concentrations of tanshinol, using 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, 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 activity of danshensu against SARS-CoV-2, and confirm that danshensu has the ability of resisting SARS-CoV-2 infection, and simultaneously has the function of inhibiting SARS-CoV-2 from entering target cells, and provide an application of danshensu in preparing anti-new type 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 tanshinol adopted in the embodiment of the invention is purchased from Shanghai ceramic Biotechnology limited company, and the purity is more than 99%.

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 single luciferase assay kit used in the examples of the present invention was purchased from PROMEGA, usa and includes cell lysate and luciferase reaction substrate.

Pharmacological experiment part

Example 1 assay of inhibitory Activity of Salvia miltiorrhiza in vitro against SARS-CoV-2

1. Drug inhibition activity assay:

1) taking Vero-E6 cells in logarithmic growth phase, 3 x 10^5 cells/well, inoculating in 48-well plate, 37 deg.C, 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 drug concentration was set at 50 μ M (solvent in DMSO), drug was diluted three-fold, 3 multiple wells per concentration, for a total of 9 drug gradients (50, 16.7, 5.56, 1.85, 0.62, 0.21, 0.07, 0.023, 0.008 μ M); solvent dimethyl sulfoxide (DMSO) is set as a control group, the control group is diluted by DMEM medium containing 2% fetal bovine serum in total volume, and the dimethyl sulfoxide with the same volume is given to 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 incubation was continued at 37 ℃ for 1 h. The infected supernatant was removed well and the cells were washed once with 200. mu.l PBS. 200 mul of culture medium containing the drug at the corresponding concentration is added into the wells again, the culture is continued for 24h, and 150 mul of cell culture supernatant is collected for testing. Viral copy number was determined using qRT-PCR.

4) For the specific operation of Viral RNA Extraction, Takara MiniBEST Viral RNA/DNA Extraction Kit (Code No. 9766):

a. splitting the virus: mu.l of PBS (pH7.4) was added to 150. mu.l of the cell culture supernatant to make up to 200. mu.l. Add 200. mu.l VGB buffer, 20. mu.l Proteinase K and 1.0. mu.l Carrier RNA, mix well and lyse well in a 56 ℃ water bath for 10 minutes. Add 200. mu.l absolute ethanol to the lysate, suck well and mix well.

b. Column passing: 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. Washing 1: mu.l of RWA buffer was added to Spin Column, centrifuged at 12,000rpm for 1 minute, and the filtrate was discarded.

d. And (3) washing 2: mu.l of RWB buffer was added to Spin Column, centrifuged at 12,000rpm for 1 minute, and the filtrate was discarded. (RWB buffer to which a specified volume of 100% ethanol had been added). RWB buffer was added around the wall of the Spin Column.

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. And (3) elution: 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.

5) Specific procedures for reverse transcription of viral RNA were described in Takara PrimeScriptTM RT reagent Kit with gDNA Eraser (Code No. RR047A):

a. removing the genomic DNA in the eluate: the reaction system is prepared on ice according to the following components

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:

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

6) qPCR assay virus copy number: reference is made to Takara TBPremix Ex Taq^TMII (TliRNaseH Plus, Code, No. RR820A) (Standard Curve method: RBD plasmid of known copy number as standard, specific primer targeting RBD). The reaction system was prepared on ice as follows:

reagent	Volume (μ l)
		TB Green Premix Ex Taq II(Tli RNaseH Plus)(2X)	10
Forward Primer(10μM)	1
		Reverse Primer(10μM)	1
ROX Reference Dye(50X)	0.4
		cDNA template	1
Sterilized water	6.6
		Total volume	20

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: 2)

And (3) computer detection: ABI7500 quantitative PCR instrument

Pre-denaturation: 95 ℃, 30 seconds, 1 cycle; and (3) PCR amplification: at 95 ℃, 5 seconds, 40 cycles; annealing: 30-34 seconds at 60 ℃; and (6) recording.

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 tanshinol (DSS) acting on SARS-CoV-2 activity₅₀It was 1.07. mu.M.

Example 2 assay of the inhibitory Activity of Salvianic acid A on the entry of the SARS-CoV-2S protein pseudovirus

1. The method comprises the following steps:

1) SARS-CoV-2S protein pseudovirus package:

HEK-293T cells in logarithmic growth phase 4 x 10^ 5/ml, 2ml per well were seeded in 6-well plates. 37 ℃ and 5% CO₂Cell cultureThe culture was carried out in a chamber for 24 hours. Replacing fresh culture medium half an hour before transfection, respectively preparing plasmid diluent and transfection reagent (PolyJet) diluent by adopting 100 mul of blank DMEM culture medium, wherein the preparation proportion of each well is as follows (plasmid DNA needs to be extracted by adopting 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. The PolyJet dilutions were added to the plasmid dilutions and mixed well, incubated for 10 min at room temperature, and added well to HEK-293T cells. Culturing at 37 deg.C for 48 hr, collecting supernatant, centrifuging at 4000rpm for 10 min, and filtering with 0.45 μm sterile filter head to obtain SARS-CoV-2 pseudovirus.

2) Pseudovirus inhibition experiments:

293T cells (293T/ACE2) overexpressing the SARS-CoV-2 receptor ACE2 in logarithmic growth phase were plated evenly in 96 well plates at 1 × 10^ 4/well. Cultured in a cell culture chamber at 37 ℃ for 24 hours.

The initial concentration of the drug is set to 40 μ M, and 9 concentration gradients are diluted 2 times by volume in DMEM medium containing 2% fetal calf serum in total volume, which are 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125 and 0.15625 μ M respectively. The DMSO solvent control was set up at 60 μ l per well volume, 3 replicates per concentration. 60 μ l of pseudovirus solution was added to the diluted drug, allowed to act at room temperature for 30 minutes, and 100 μ l/well was added to ACE2/293T cells and cultured at 37 ℃ for 48 hours. The medium was removed and the cells were washed once with 100. mu.l/well sterile PBS (pH7.4), 50. mu.l of 1X cell lysate was added to each well and lysed with shaking at room temperature for 15 minutes. Transferring 40 mul/hole cracking supernatant to a 96-hole white enzyme label plate, adding isovolumic 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 danshensu for inhibiting virus adsorption according to the fluorescence value. According to the fluorescence valueCalculating the inhibition rate according to the corresponding relationship with the drug concentration, drawing a curve, and calculating the half inhibition concentration IC of tanshinol₅₀。

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 inhibition concentration IC of tanshinol (DSS) for inhibiting SARS-CoV-2S protein pseudovirus from entering target cells₅₀It was 2.70. mu.M.

Example 3 cytotoxicity assay of Salvianic acid A

1. The method comprises the following steps:

1) cell inoculation:

Vero-E6, 293T-ACE2 cells in logarithmic growth phase were adjusted to 1 × 10^4 cells/well, seeded in 96-well plates at 100 μ L/well, and cultured overnight.

2) Designing the concentration of the medicine:

before administration, 9 concentration gradients were diluted 2-fold in DMEM medium containing 2% fetal bovine serum in total volume, the initial concentration of the drug was set at 200. mu.M (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125. mu.M), 100. mu.L of the diluted drug per well was added to Vero-E6 and 293T-ACE2 cells in a 96-well plate 1), and the final volume of each well was 200. mu.L. 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;

salvianic acid A (DSS) has no obvious toxic effect on Vero-E6 cells (figure 3) and 293T/ACE2 cells (figure 4) within 200 mu M 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 danshensu or pharmaceutically acceptable salt thereof in preparing antiviral drug

<130> CP120010255C

<160> 2

<170> PatentIn version 3.3

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<211> 21

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caatggttta acaggcacag g 21

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ctcaagtgtc tgtggatcac g 21