CN113230261A - Application of dihydrotanshinone I or pharmaceutically acceptable salt thereof in preparation of anti-coronavirus medicines - Google Patents
Application of dihydrotanshinone I or pharmaceutically acceptable salt thereof in preparation of anti-coronavirus medicines Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- Life Sciences & Earth Sciences (AREA)
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- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an application of dihydrotanshinone I or a pharmaceutically acceptable salt thereof in preparing an anti-coronavirus medicament. The invention discovers the application of the dihydrotanshinone I or the pharmaceutically acceptable salt thereof in preparing the anti-coronavirus medicament for the first time, expands the application range of the dihydrotanshinone I, and provides certain feasible basis and new medicament for treating novel coronavirus pneumonia by clinical traditional Chinese medicines.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of dihydrotanshinone I or pharmaceutically acceptable salt thereof in preparing an anti-coronavirus medicine.
Background
Coronaviruses are single-stranded positive-strand ribonucleic acid (RNA) viruses with envelope, have a genome size of 27-32kb, and are the largest viruses among the RNA viruses known at present. The novel Coronavirus pneumonia (Covid-19) pathogen was isolated and identified as "Severe Acute Respiratory Syndrome Coronavirus 2" (SARS-CoV-2). SARS-CoV-2 spike protein (S protein) is a key protein of a novel coronavirus infected cell, plays an important role in aspects of host tropism, virulence and the like of the virus, and is an important target for searching novel inhibitors, antibody medicaments and vaccine development.
Salvia miltiorrhiza is the dried root and rhizome of Salvia miltiorrhiza bge, which has been studied and shown to have various pharmacological effects, such as anticancer, anti-inflammatory, estrogen-like activity, cardiovascular protection, and some positive bacteria resistance, and is a common medicine for clinical treatment of coronary heart disease and ischemic cerebrovascular disease. The Saviae Miltiorrhizae radix mainly comprises tanshinone, salvianolic acid, volatile oil and inorganic elements. The nature of the fat-soluble tanshinone compounds is a kind of fat-soluble diterpene quinone compounds, and the tanshinone compounds separated at present mainly comprise more than 10 tanshinone monomers such as dihydrotanshinone I, cryptotanshinone, isocytotanshinone, tanshinone A, tanshinone IIA, tanshinone IIB and the like. No report on the application of tanshinone compounds in treating Xinguan diseases is found.
Disclosure of Invention
The invention aims to provide a novel anti-coronavirus drug.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides an application of dihydrotanshinone I or pharmaceutically acceptable salt thereof in preparing anti-coronavirus medicines, wherein the molecular formula of the dihydrotanshinone I is C18H14O3Molecular weight of 278.3, and structural formula
In another aspect, the invention provides an application of dihydrotanshinone I or a pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting coronavirus from entering a target cellThe molecular formula of the dihydrotanshinone I is C18H14O3Molecular weight of 278.3, and structural formula
The invention also provides application of the dihydrotanshinone I analogue or the pharmaceutically acceptable salt thereof in preparing the anti-coronavirus medicament.
In another aspect, the invention provides an application of a dihydrotanshinone I analogue or a pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting a coronavirus from entering a target cell.
In still another aspect, the present invention provides an anti-coronavirus pharmaceutical composition comprising dihydrotanshinone I or a pharmaceutically acceptable salt thereof as an active substance, wherein the dihydrotanshinone I has a molecular formula of C18H14O3Molecular weight of 278.3, and structural formula
In still another aspect, the present invention provides a pharmaceutical composition for inhibiting entry of coronavirus into a target cell, comprising dihydrotanshinone I having a molecular formula of C or a pharmaceutically acceptable salt thereof as an active substance18H14O3Molecular weight of 278.3, and structural formula
In still another aspect, the present invention provides an anti-coronavirus pharmaceutical composition comprising a dihydrotanshinone I analog or a pharmaceutically acceptable salt thereof as an active substance.
In still another aspect of the present invention, there is provided a pharmaceutical composition for inhibiting entry of coronavirus into a target cell, which comprises a dihydrotanshinone I analog or a pharmaceutically acceptable salt thereof as an active substance.
Further, the dihydrotanshinone I analogue comprises cryptotanshinone and tanshinone A.
Further, the coronavirus includes HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Severe acute respiratory syndrome coronavirus SARS-CoV, Severe acute respiratory syndrome coronavirus 2 SARS-CoV-2, and Zhongdong respiratory syndrome coronavirus MERS-CoV; preferably, the coronavirus is SARS-CoV-2.
Further, the pharmaceutical composition is an oral preparation or an injection preparation.
Further, the pharmaceutical composition is an oral capsule, a tablet, an oral liquid or an injection.
The invention has the following beneficial effects:
the invention discovers the application of the dihydrotanshinone I, the dihydrotanshinone I analogue or the pharmaceutically acceptable salt thereof in preparing the anti-coronavirus medicament for the first time, the application expands the application range of the dihydrotanshinone I and the dihydrotanshinone I analogue, and provides certain feasible basis and new medicament for treating novel coronavirus pneumonia by using the traditional Chinese medicine clinically.
Experiments show that the Dihydrotanshinone I and the Dihydrotanshinone I analogue can effectively inhibit SARS-CoV-2 on in-vitro cultured cell Vero-E6, and the half effective concentration EC of the Dihydrotanshinone I and the Dihydrotanshinone I analogue is used as an antiviral drug500.64. mu.M; application of dihydrotanshinone I in inhibiting SARS-CoV-2 entry stage, inhibiting SARS-CoV-2 pseudovirus infection of 293T/ACE2 cell, and inhibiting half inhibitory concentration IC500.68 μ M; in addition, Tanshinone A (T-A) inhibits EC of SARS-CoV-2502.98. mu.M; cryptotanshinone (Cry-T) EC for inhibiting SARS-CoV-250It was 1.46. mu.M. The dihydrotanshinone I, tanshinone A and cryptotanshinone have no obvious cytotoxicity 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 dihydrotanshinone I (DHT) concentration-dependent inhibition of SARS-CoV-2 in example 1 of the present invention, in which the abscissa represents the concentration of dihydrotanshinone I and the ordinate represents the inhibition rate of dihydrotanshinone I against SARS-CoV-2 with respect to the solvent group, and calculation was performed based on the inhibition rateHalf effective concentration EC for inhibiting SARS-CoV-2 by dihydrotanshinone I50The value is obtained.
FIG. 2 is a graph showing the inhibition rate of tanshinone A (T-A) against SARS-CoV-2 at different concentrations in mutexample 2 of the present invention, in which the abscissa represents the concentration of tanshinone A and the ordinate represents the inhibition rate of tanshinone A against SARS-CoV-2 using the solvent group as a control, and the half effective concentration EC of tanshinone A against SARS-CoV-2 was calculated from the inhibition rates50The value is obtained.
FIG. 3 is EC of cryptotanshinone (Cry-T) for inhibiting SARS-CoV-2 in example 3 of the present invention50A value curve chart, wherein the abscissa represents the concentration of cryptotanshinone, the ordinate represents the inhibition rate of cryptotanshinone on SARS-CoV-2 by using solvent group as control, and the half effective concentration EC of cryptotanshinone for inhibiting SARS-CoV-2 is calculated according to the inhibition rate50The value is obtained.
FIG. 4 is a graph showing the inhibition rate of dihydrotanshinone I (DHT) against the entry of SARS-CoV-2 pseudovirus into target cells at different concentrations in example 4 of the present invention, wherein the abscissa represents the concentration of dihydrotanshinone I, and the ordinate represents the inhibition rate of dihydrotanshinone I against the entry of SARS-CoV-2 pseudovirus using solvent group as control, and the half-inhibitory concentration IC of dihydrotanshinone I against the entry of SARS-CoV-2 pseudovirus is determined50The value is obtained.
FIG. 5 is a graph showing the survival rate of dihydrotanshinone I (DHT) versus Vero-E6 cells in example 5 of the present invention, in which the abscissa represents the concentration of dihydrotanshinone I and the ordinate represents the percentage of cells surviving after administration of different concentrations of dihydrotanshinone I to Vero-E6 cells in the control solvent group.
FIG. 6 is a graph showing the survival rate of dihydrotanshinone I (DHT) versus 293T/ACE2 cells in example 5, wherein the abscissa represents the concentration of dihydrotanshinone I and the ordinate represents the percentage of cell survival of 293T/ACE2 cells after administration of different concentrations of dihydrotanshinone I, in which the control group of solvents was used.
FIG. 7 is a graph showing the survival rate of 293T/ACE2 cells in combination with tanshinone A (T-A) in mutexample 5, wherein the abscissa represents the tanshinone A concentration and the ordinate represents the percentage of cell survival of 293T/ACE2 cells after administration of different concentrations of tanshinone A, in contrast to the solvent group.
FIG. 8 is a graph showing the survival rate of 293T/ACE2 cells in accordance with cryptotanshinone (Cry-T) in example 5 of the present invention, wherein the abscissa represents the concentration of cryptotanshinone and the ordinate represents the percentage of cell survival of 293T/ACE2 cells after administration of different concentrations of cryptotanshinone in the case of solvent group as 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 evaluate the activity of dihydrotanshinone I and its analogs against SARS-CoV-2 and confirm that dihydrotanshinone I and its analogs have the ability to resist SARS-CoV-2 infection mainly by constructing SARS-CoV-2 live virus and SARS-CoV-2 pseudovirus in vitro cell infection models. Meanwhile, the dihydrotanshinone I has the function of inhibiting SARS-CoV-2 from entering target cells, and provides application of the dihydrotanshinone I and the analog thereof in preparing medicaments for resisting novel coronavirus.
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 dihydrotanshinone I, the tanshinone A and the cryptotanshinone adopted in the embodiment of the invention are purchased from Shanghai pottery 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 a cell lysate and a luciferase reaction substrate.
Pharmacological experiment part
Example 1 measurement of inhibitory Activity of dihydrotanshinone I against SARS-CoV-2 in vitro.
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 10 μ M (solvent DMSO), drug was diluted three-fold, 3 multiple wells per concentration, for a total of 6 drug gradients (10, 3.33, 1.11, 0.37, 0.12, 0.04 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) in 48-well plates, 100. mu.l of diluted drug was added to each well of the experimental group, 100. mu.l of diluted DMSO was added to the control group, and incubation was performed at 37 ℃ for 1 hour.
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 Spin Column 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. 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:
| reagent | Volume (μ l) |
| Reaction solution of |
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.
6) qPCR assay virus copy number: reference is made to Takara TB
Premix Ex TaqTMII (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 |
| |
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 dihydrotanshinone I (DHT) acting on SARS-CoV-2 activity50It was 0.64. mu.M.
Example 2 assay of inhibitory Activity of tanshinone A against SARS-CoV-2 in vitro.
1. Drug inhibitory activity assay methods:
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 100 μ M (solvent DMSO), drug was diluted three-fold, 3 multiple wells per concentration, for a total of 6 drug gradients (100, 33.33, 11.11, 3.70, 1.23, 0.41 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) 4), 5), 6), 7) are the same as in example 1.
2. As a result: as shown in fig. 2;
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 tanshinone A (T-A) acting on SARS-CoV-2 activity50It was 2.98. mu.M.
Example 3 detection of inhibitory Activity of Cryptodanthrone on SARS-CoV-2 in vitro.
1. Drug inhibitory activity assay methods:
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 100 μ M (solvent DMSO), drug was diluted three-fold, 3 multiple wells per concentration, for a total of 6 drug gradients (100, 33.33, 11.11, 3.70, 1.23, 0.41 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) 4), 5), 6), 7) are the same as in example 1.
2. As a result: as shown in fig. 3;
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. Then prism8.0 soft tissue is applied according to the inhibition rate of drug treatment groups with different concentrationsFitting a drug inhibition rate curve, and calculating the half effective concentration EC of cryptotanshinone (Cry-T) acting on SARS-CoV-2 activity50It was 1.46. mu.M.
Example 4 measurement of inhibitory Activity of dihydrotanshinone I against entry of SARS-CoV-2 pseudovirus.
1. The method comprises the following steps:
1) SARS-CoV-2 pseudovirus packaging:
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. Replacing fresh culture medium half an hour before transfection, preparing plasmid diluent and transfection reagent (PolyJet) diluent by respectively adopting 100 mul of blank DMEM culture medium, wherein the preparation proportion of each hole 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-S 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 dihydrotanshinone I is set to be 2.5 mu M, and 8 concentration gradients which are 2 times diluted by adopting DMEM culture medium containing 2 percent of fetal calf serum in total volume are respectively 2.50, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039 and 0.020 mu M. Pore volume 60 mu per porel, 3 replicates per concentration, DMSO solvent control was set up. 60. mu.l of pseudovirus solution was added to the diluted drug, allowed to act at room temperature for 30 minutes, and 100. mu.l/well was added to 293T/ACE2 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 mu l/hole of the cracking supernatant into a 96-hole white enzyme label plate, adding an isovolumic diluted luciferase substrate according to the specification of a single luciferase detection reagent kit, immediately carrying out enzyme label instrument detection on a fluorescence value, and judging the activity of dihydrotanshinone I for inhibiting virus adsorption entry 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 dihydrotanshinone I50。
2. As a result: as shown in fig. 4;
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 dihydrotanshinone I (DHT) for inhibiting SARS-CoV-2 pseudovirus from entering target cells50It was 0.68. mu.M.
Example 5 cytotoxicity assay of Dihydrotanshinone I, tanshinone A, cryptotanshinone
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:
Vero-E6 cells: diluting with DMEM medium containing 2% fetal calf serum by 3 times to obtain 8 concentration gradients before administration, wherein the initial concentration of dihydrotanshinone I is 50 μ M (50, 16.67, 5.56, 1.85, 0.62, 0.21, 0.07, 0.02 μ M); setting the initial concentration of tanshinone A at 100 μ M (100, 33.33, 11.11, 3.70, 1.23, 0.41, 0.13, 0.05 μ M); the initial concentration of cryptotanshinone is set to 100 μ M (100, 33.33, 11.11, 3.70, 1.23, 0.41, 0.13, 0.05 μ M); 100. mu.L of the diluted drug per well was added to Vero-E6 cells in 96-well plates in 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.
293T/ACE2 cells: before administration, DMEM medium containing 2% fetal calf serum in total volume is diluted by 2 times to 8 concentration gradients, and initial concentration of dihydrotanshinone I is set to 10 μ M (10, 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08 μ M). 100. mu.L of the diluted drug per well was added to 293T/ACE2 cells in 96-well plates in 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 to the control group.
2. As a result:
as shown in FIG. 5, dihydrotanshinone I (DHT) had no significant toxic effect on Vero-E6 cells in the effective concentration range of 10 μ M.
As shown in FIG. 6, dihydrotanshinone I (DHT) has no significant toxic effect on 293T/ACE2 cells within the effective concentration range of 2.5. mu.M.
As shown in FIG. 7, tanshinone A (T-A) has no significant toxic effect on Vero-E6 cells in the effective concentration range of 100 μ M.
As shown in FIG. 8, cryptotanshinone A (Cry-T) had no significant toxic effect on Vero-E6 cells in the effective concentration range of 100. mu.M.
The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent changes to the technical solution of the present invention by a person skilled in the art after reading the present specification are covered by the claims of the present invention.
SEQUENCE LISTING
<110> southern medical university
Application of dihydrotanshinone I or pharmaceutically acceptable salt thereof in preparation of anti-coronavirus medicines
<130> CP120010859C
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence
<400> 1
caatggttta acaggcacag g 21
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence
<400> 2
ctcaagtgtc tgtggatcac g 21
Claims (10)
1. Application of dihydrotanshinone I or pharmaceutically acceptable salt thereof in preparation of anti-coronavirus medicines, wherein the molecular formula of dihydrotanshinone I is C18H14O3Molecular weight of 278.3, and structural formula
2. Application of dihydrotanshinone I or pharmaceutically acceptable salt thereof in preparing medicine for inhibiting coronavirus from entering target cell, wherein the molecular formula of dihydrotanshinone I is C18H14O3Molecular weight of 278.3, and structural formula
3. Application of dihydrotanshinone I analogue or its pharmaceutically acceptable salt in preparing medicine for resisting coronavirus is provided.
4. Application of dihydrotanshinone I analogue or its pharmaceutically acceptable salt in preparing medicine for inhibiting coronavirus from entering target cell is provided.
5. An anti-coronavirus pharmaceutical composition comprising a dihydrotanshinone I having a molecular formula of C or a pharmaceutically acceptable salt thereof as an active substance18H14O3Molecular weight of 278.3, and structural formula
6. A pharmaceutical composition for inhibiting entry of coronavirus into a target cell, characterized by comprising dihydrotanshinone I as an active substance having a molecular formula of C or a pharmaceutically acceptable salt thereof18H14O3Molecular weight of 278.3, and structural formula
7. An anti-coronavirus pharmaceutical composition characterized by containing a dihydrotanshinone I analog or a pharmaceutically acceptable salt thereof as an active substance.
8. A pharmaceutical composition for inhibiting entry of coronavirus into a target cell, which comprises a dihydrotanshinone I analog or a pharmaceutically acceptable salt thereof as an active substance.
9. The use as claimed in any one of claims 3 to 4 or the pharmaceutical composition as claimed in any one of claims 7 to 8, wherein the dihydrotanshinone I analogue comprises tanshinone A, cryptotanshinone.
10. The use of any one of claims 1 to 4 or the pharmaceutical composition of any one of claims 5 to 8, wherein the coronavirus comprises HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV-2, MERS-CoV; preferably, the coronavirus is SARS-CoV-2.
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| JP2022568650A JP2023527706A (en) | 2020-05-18 | 2021-05-17 | Use of danshen active ingredient or a pharmaceutically acceptable salt thereof in the manufacture of an antiviral drug |
| PCT/CN2021/094045 WO2021233239A1 (en) | 2020-05-18 | 2021-05-17 | Application of active ingredient of root of ligulilobe sage or pharmaceutically acceptable salt thereof in preparing antiviral drug |
| EP21808105.7A EP4154878A4 (en) | 2020-05-18 | 2021-05-17 | Application of active ingredient of root of ligulilobe sage or pharmaceutically acceptable salt thereof in preparing antiviral drug |
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| CN113679726A (en) * | 2020-05-18 | 2021-11-23 | 上海科技大学 | Application of salvia miltiorrhiza extract and quinone compounds in resisting coronavirus |
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