阅读说明：本技术 丹酚酸c或其药物可接受盐在制备抗病毒的药物中的应用 (Application of salvianolic acid C or pharmaceutically acceptable salt thereof 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：本发明公开了一种丹酚酸C或其药物可接受盐在制备抗病毒的药物中的应用。本发明首次提出丹酚酸C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,该应用扩大了丹酚酸C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 salvianolic acid C or a pharmaceutically acceptable salt thereof in preparing antiviral drugs. The invention firstly provides the application of the salvianolic acid C or the pharmaceutically acceptable salt thereof in preparing antiviral medicaments, provides a new treatment means for new crown infectors with chronic basic diseases, enlarges the application range of the salvianolic acid C, and provides a new medicament for inhibiting viruses, particularly SARS-CoV-2 under the condition of global new crown epidemic.)

1. Application of salvianolic acid C or its pharmaceutically acceptable salt in preparing antiviral drugs, wherein the molecular formula of salvianolic acid C is C₂₆H₂₀O₁₀Molecular weight of 492.43, and structural formula

2. Application of salvianolic acid C or pharmaceutically acceptable salt thereof in preparing medicament for inhibiting virus from entering target cells, wherein the molecular formula of salvianolic acid C is C₂₆H₂₀O₁₀Molecular weight of 492.43, 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 as an active ingredient salvianolic acid C of the formula C or a pharmaceutically acceptable salt thereof₂₆H₂₀O₁₀Molecular weight of 492.43, and structural formula

6. A pharmaceutical composition for inhibiting viral entry into a target cell, comprising as an active ingredient salvianolic acid C of the formula C or a pharmaceutically acceptable salt thereof₂₆H₂₀O₁₀Molecular weight of 492.43, 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 salvianolic acid C or a pharmaceutically acceptable salt thereof in preparing an antiviral drug.

Background

Recently, the new coronavirus pneumonia (COVID-19) caused by SARS-CoV-2 invasion has been outbreak worldwide, and becomes the world's leading public health threat due to its extremely high transmission rate and ultra-long latent period. 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. Entry of SARS-CoV-2 into the host cell is mediated by the transmembrane Spike S glycoprotein (S), where the S protein forms a homotrimer protruding from the virus surface. The S protein comprises two functional subunits, S1 and S2, where S1 is responsible for binding to host cell receptors and the S2 subunit is responsible for viral membrane and cell membrane fusion.

It is currently believed that the S2 protein has at least three conformations: the native conformation before fusion, the intermediate conformation before hairpin and the hairpin conformation after fusion, through these different conformations drive the fusion of the virus with the target cell. Therefore, the S protein inhibitor can prevent viruses from entering host cells, and the polypeptide fusion mediated by the S2 subunit can be used as a target point for screening antiviral drugs. At present, few compounds aiming at the target point are available, no drug is approved to be on the market, and no effective entry inhibitor targeting SARS-CoV-2S 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. The water-soluble phenolic acid components of the salvia miltiorrhiza mainly have the antioxidation effect, the content of the salvianolic acid C in the salvia miltiorrhiza is lower, and the pharmacological action research is less. Salvianolic acid C (Salvinolic acid C) has strong pharmacological action in the aspects of oxidation resistance, liver injury resistance, tumor resistance, thrombus resistance and the like. Free radical scavenging activity of salvianolic acid C was disclosed in patent CN106596432A by Wangsheng et al, 2016; application of salvianolic acid C in preparation of anti-stroke medicines in CN109985033A was disclosed in 2017 by Duguanhua et al; the application of salvianolic acid C in preparing medicines for protecting ischemic brain tissue damage is disclosed in patent CN110101693A of Fanccellover et al in 2019; in 2019, Tanghong Ching et al in patent CN109820889A disclose the application of salvianolic acid C in preparing protein tyrosine phosphatase 1B inhibitor and medicines for preventing and/or treating type 2 diabetes, and there is no application in preventing or treating 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 salvianolic acid C or its pharmaceutically acceptable salt in preparing antiviral drugs, wherein the molecular formula of the salvianolic acid C is C₂₆H₂₀O₁₀Molecular weight of 492.43, and structural formula

In another aspect of the present invention, there is provided a use of salvianolic acid C having a molecular formula of C or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting a virus from entering a target cell₂₆H₂₀O₁₀Molecular weight of 492.43, and structural 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 salvianolic acid C of the molecular formula C or a pharmaceutically acceptable salt thereof as an active ingredient₂₆H₂₀O₁₀Molecular weight of 492.43, and structural formula

In still another aspect, the present invention provides a pharmaceutical composition for inhibiting viral entry into a target cell, comprising salvianolic acid C of the molecular formula C or a pharmaceutically acceptable salt thereof as an active ingredient₂₆H₂₀O₁₀Molecular weight of 492.43, 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 salvianolic acid C or the pharmaceutically acceptable salt thereof in preparing antiviral medicaments, provides a new treatment means for new crown infectors with chronic basic diseases, enlarges the application range of the salvianolic acid C, and provides a new medicament for inhibiting viruses, particularly SARS-CoV-2 under the condition of global new crown epidemic.

The salvianolic acid C is used as an antiviral drug, and experiments show that the salvianolic acid C inhibits the half effective concentration EC of SARS-CoV-2 activity on in-vitro cultured cell Vero-E6₅₀3.41 μ M; salvianolic acid C is used for inhibiting SARS-CoV-2 entry stage, inhibiting SARS-CoV-2S protein pseudovirus infection overexpression 293T/ACE2 cell, and inhibiting half effective inhibitory concentration IC₅₀5.90. mu.M; the salvianolic acid C has no obvious cytotoxicity in an effective concentration range. Therefore, can be used for preparing anti-SARS-CoV-2 medicaments.

Drawings

FIG. 1 is a graph showing the inhibition rate of salvianolic acid C (Sal-C) inhibiting SARS-CoV-2 at different concentrations in example 1, in which the abscissa represents the concentration of salvianolic acid C and the ordinate represents the inhibition rate of salvianolic acid C on SARS-CoV-2 with respect to solvent group as control, and the half effective concentration EC of salvianolic acid C inhibiting SARS-CoV-2 is calculated from the inhibition rate₅₀The value is obtained.

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

FIG. 3 is a graph showing the survival rate of Vero-E6 cells as target cells against salvianolic acid C (Sal-C) in example 3, wherein the abscissa represents the concentration of salvianolic acid C and the ordinate represents the percentage of cells surviving after administration of Vero-E6 cells at different concentrations of salvianolic acid C as a control in a solvent group.

FIG. 4 is a graph showing the survival rate of 293T/ACE2 cells as target cells against salvianolic acid C (Sal-C) in example 3, wherein the abscissa represents the concentration of salvianolic acid C and the ordinate represents the percentage of cell survival of 293T/ACE2 cells after administration of different concentrations of salvianolic acid C as a control of solvent.

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 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 models to evaluate the anti-SARS-CoV-2 activity of salvianolic acid C, and confirm that salvianolic acid C has the ability of anti-SARS-CoV-2 infection, and simultaneously has the effect of inhibiting SARS-CoV-2 from entering target cells, and provide an application of salvianolic acid C in preparing anti-new coronavirus drugs.

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 salvianolic acid C 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 SARS-CoV-2S pseudovirus packaging skeleton plasmid pNL4-3.Luc. R-E-is collected, identified and stored for the unit, and the disclosed SARS-CoV-2S protein full-length core plasmid pcDNA3.1-SARS-CoV-2-Sipke is offered by professor Lu Dai of Shanghai Fudan 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.

Pharmacological experiment part

Example 1 detection of inhibitory Activity of Salvianolic acid C on SARS-CoV-2 in vitro

1. Determination of SARS-CoV-2 inhibition by drugs

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. The initial concentration of the drug is set to be 50 mu M (the solvent is DMSO), the drug is diluted by three times, each concentration of the drug is provided with 3 multiple wells, 9 drug gradients are totally set, the concentration is respectively 50, 16.7, 5.56, 1.85, 0.62, 0.21, 0.07, 0.023 and 0.008 mu M, the solvent is dimethyl sulfoxide (DMSO) and is used as a control group, and the control group is diluted by DMEM culture medium containing 2 percent of fetal calf serum in total volume and is given with the same volume of dimethyl sulfoxide. 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 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.

5) Specific procedures for Viral RNA Extraction (see Takara MiniBEST Viral RNA/DNA Extraction 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 EraserCode 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

Reaction procedure: 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

Reaction procedure: 15min at 37 ℃; 85 ℃ for 5 sec.

7) 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 was used as a standard, and specific primers were targeted to RBD). The reaction solution 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)

Detection was done 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 salvianolic acid C (Sal-C) acting on SARS-CoV-2₅₀It was 3.41. mu.M.

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

1. The method comprises the following steps:

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

HEK-293T cells in logarithmic growth phase 4 x 10^5Each well was inoculated in 6 well plates at 2 ml/well. 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:

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 was set at 20 μ M, and 9 concentration gradients (20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.07813 μ M) were diluted 2-fold in DMEM medium containing 2% fetal bovine serum in total volume before administration, 60 μ l per well, 3 duplicate wells per concentration, and DMSO solvent control was 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 ℃. 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 96-hole white enzyme label plate, adding isovolumic diluted luciferase substrate according to the specification of single luciferase detection kit, and immediately detecting fluorescence value and root by enzyme labelAnd judging the activity of the danshensu 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 to calculate the semi-inhibitory concentration IC of the drug₅₀。

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 the salvianolic acid C (Sal-C) for inhibiting SARS-CoV-2S protein pseudovirus from entering target cells₅₀It was 5.90. mu.M.

Example 3 cytotoxicity assay for Salvianolic acid C

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 with an initial concentration of 200. mu.M (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125. mu.M) and 100. mu.L of the diluted drug per well was added to Vero-E6 and 293T-ACE2 cells in 96-well plates 1) to 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 C (Sal-C) has no obvious toxic effect on Vero-E6 cells (figure 3) and 293T/ACE2 cells (figure 4) in 200 μ 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

Application of <120> salvianolic acid C or pharmaceutically acceptable salt thereof in preparing antiviral drugs

<130> CP120010257C

<160> 2

<170> PatentIn version 3.3

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