Application of salvianolic acid B or its pharmaceutically acceptable salt in preparing anti-SARS-CoV-2 medicine
阅读说明：本技术 丹酚酸B或其药物可接受盐在制备抗SARS-CoV-2的药物中的应用 (Application of salvianolic acid B or its pharmaceutically acceptable salt in preparing anti-SARS-CoV-2 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酚酸B或其药物可接受盐在制备抗SARS-CoV-2的药物中的应用。本发明首次提出丹酚酸B或其药物可接受盐在制备抗SARS-CoV-2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B的使用范围,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 salvianolic acid B or a pharmaceutically acceptable salt thereof in preparing a medicine for resisting SARS-CoV-2. The invention firstly provides the application of the salvianolic acid B or the pharmaceutically acceptable salt thereof in preparing the anti-SARS-CoV-2 medicament, provides a new treatment means for new crown infectors with chronic basic diseases, enlarges the application range of the salvianolic acid B, and particularly provides a new medicament for treating pneumonia (COVID-19) caused by new crown virus infection under the condition of global SARS-CoV-2 epidemic.)
1. Application of salvianolic acid B or its pharmaceutically acceptable salt in preparing antiviral drugs, wherein the molecular formula of salvianolic acid B is C36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
2. Salvianolic acid B or its pharmaceutically acceptable saltApplication of salt in preparation of medicine for inhibiting virus from entering target cell, wherein salvianolic acid B has molecular formula of C36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
3. Application of salvianolic acid B or pharmaceutically acceptable salt thereof in preparation of medicines for inhibiting virus S protein-mediated virus-cell fusion, wherein the molecular formula of salvianolic acid B is C36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
4. Application of salvianolic acid B or pharmaceutically acceptable salt thereof in preparing mediated medicines for inhibiting formation of six-helix structure in fusion regions HR1 and HR2 of S2 subunit of virus S protein, wherein the molecular formula of salvianolic acid B is C36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
5. An antiviral pharmaceutical composition comprising as an active ingredient salvianolic acid B of the formula C or a pharmaceutically acceptable salt thereof36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
6. A pharmaceutical composition for inhibiting SARS-CoV-2 entry into a target cell, comprising as an active ingredient salvianolic acid B of formula C or a pharmaceutically acceptable salt thereof36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
7. A pharmaceutical composition for inhibiting virus S protein-mediated virus-cell fusion, comprising salvianolic acid B or a pharmaceutically acceptable salt thereof as an active agent, wherein salvianolic acid B has the formula C36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
8. A pharmaceutical composition for inhibiting the formation of six-helix structure in the fusion regions HR1 and HR2 of the S2 subunit of viral protein, comprising salvianolic acid B or its pharmaceutically acceptable salt as active ingredient, wherein the molecular formula of said salvianolic acid B is C36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
9. The pharmaceutical composition according to any one of claims 5 to 8, 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 injectable powder, 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 B or a pharmaceutically acceptable salt thereof in preparing a medicine for resisting SARS-CoV-2.
Background
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 to mediate viral entry, and the S2 subunit is responsible for viral and cell membrane fusion. 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 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 component of salvia miltiorrhiza mainly has an antioxidant effect, and salvianolic acid B (Salvianolic acid B) is one of the natural products with the strongest antioxidant activity at present, can remove oxygen free radicals and inhibit lipid peroxidation, and can also inhibit platelet aggregation and thrombosis and has important pharmacological effects on organs such as heart, brain, liver, kidney and the like. In 2013, the application of salvianolic acid B in preparing anti-H5N 1 influenza virus medicines is disclosed in Chinese patent CN103127050A by roenza and et al, but the mechanism of the salvianolic acid B is that the salvianolic acid B can effectively inhibit the activity of RNA polymerase subunit PAN protein of the H5N1 influenza virus so as to inhibit the replication of the H5N1 influenza virus, and the PAN protein expressed by the N-terminal nucleotide sequence of the RNA polymerase subunit PA of the H5N1 influenza virus is selected as a target protein.
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:
in one aspect of the inventionProvides the application of salvianolic acid B or its pharmaceutically acceptable salt in preparing antiviral drugs, wherein the molecular formula of the salvianolic acid B is C36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
The invention also provides application of the salvianolic acid B or the pharmaceutically acceptable salt thereof in preparing a medicament for inhibiting viruses from entering target cells, wherein the molecular formula of the salvianolic acid B is C36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
In another aspect, the invention provides the use of salvianolic acid B of formula C or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting virus S protein-mediated virus-cell fusion36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
The invention further provides application of salvianolic acid B or pharmaceutically acceptable salt thereof in preparing a mediated medicament for inhibiting six-helix structure formation of fusion regions HR1 and HR2 of S2 subunit of virus S protein, wherein the molecular formula of the salvianolic acid B is C36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
In still another aspect, the present invention provides an antiviral pharmaceutical composition comprising salvianolic acid B of formula C or a pharmaceutically acceptable salt thereof as an active ingredient36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
In still another aspect, the present invention provides a pharmaceutical composition for inhibiting SARS-CoV-2 entry into a target cell, comprising salvianolic acid B of the formula C or a pharmaceutically acceptable salt thereof as an active agent36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
In another aspect, the present invention provides a pharmaceutical composition for inhibiting virus S protein-mediated virus-cell fusion, comprising salvianolic acid B of formula C or a pharmaceutically acceptable salt thereof as an active ingredient36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
The invention also relates toOn the one hand, the pharmaceutical composition for inhibiting the six-helix structure formation mediated by the fusion regions HR1 and HR2 of the S2 subunit of the virus contains salvianolic acid B or its pharmaceutically acceptable salt as an active substance, wherein the molecular formula of the salvianolic acid B is C36H30O16Molecular weight of 718.59, and structural formula
The virus includes HIV-1, SARS-CoV-2, MERS-CoV; preferably, the virus is SARS-CoV-2.
Further, the pharmaceutical composition is an injection preparation or an oral preparation;
further, the pharmaceutical composition is an injection powder injection, a tablet, a capsule, a pill or a drop pill.
The invention has the following beneficial effects:
the invention provides the application of salvianolic acid B or its pharmaceutically acceptable salt in preparing anti-SARS-CoV-2 medicine for the first time, provides a new treatment means for patients infected by novel coronavirus, especially for patients with chronic basic diseases in cardiovascular and cerebrovascular aspects such as atherosclerosis, the application expands the application range of salvianolic acid B, and provides a new medicine for inhibiting SARS-CoV-2 infection, especially for treating pneumonia (COVID-19) caused by SARS-CoV-2 infection under the condition that global new coronavirus is prevalent.
The salvianolic acid B or the pharmaceutically acceptable salt thereof is used as an antiviral drug, and experiments show that: half effective concentration EC of anti-SARS-CoV-2 activity of salvianolic acid B in vitro cultured cell Vero-E6 infection model5055.47 μ M salvianolic acid B has effect in inhibiting SARS-CoV-2 entry stage, and can inhibit SARS-CoV-2S pseudovirus activity, IC50It was 0.416. mu.M. 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 shows salvianolic acid B in example 1 of the present inventionThe inhibition rate curve chart of inhibiting SARS-CoV-2 at different concentrations, wherein the abscissa represents salvianolic acid B concentration, and the ordinate represents SARS-CoV-2 inhibition rate of salvianolic acid B compared with solvent group, and the half effective concentration EC of salvianolic acid B for inhibiting SARS-CoV-2 is calculated according to the inhibition rate50The value is obtained.
FIG. 2 is a graph showing the inhibition rate of salvianolic acid B in example 2 for inhibiting SARS-CoV-2S protein pseudovirus from entering target cells at different concentrations, wherein the abscissa represents the concentration of salvianolic acid B, and the ordinate represents the inhibition rate of salvianolic acid B in inhibiting SARS-CoV-2S protein pseudovirus from entering the target cells under control of solvent group, and the half inhibition concentration IC50 value of salvianolic acid B in inhibiting SARS-CoV-2S protein pseudovirus from entering the target cells is determined.
FIG. 3 is a schematic spectrum of scanning wavelength (abscissa) and molar circumference ratio (ordinate) curves of circular dichroism (CD for short), wherein the scanning wavelength range is 190-260 nm. The SARS-CoV-2S protein six-helix core region polypeptide HR1P, HR2P spontaneously formed alpha-helix conformation, the spectrum showed double negative peaks (SARS-CoV-2-HR1P/HR2P) at 208 and 222nm, and the curve after administration of standard 5. mu.M salvianolic acid B was SARS-CoV-2-HR1P/HR2P + Sal-B5. mu.M.
FIG. 4 is a graph showing the survival rate of Vero-E6 cells as target cells against salvianolic acid B in example 3, wherein the abscissa represents the concentration of danshensu and the ordinate represents the survival percentage of Vero-E6 cells after administration of different concentrations of salvianolic acid B in the case of solvent group as control.
FIG. 5 is a graph showing the survival rate of the target cell 293T/ACE2 with salvianolic acid B in example 3, wherein the horizontal axis represents the concentration of salvianolic acid B and the vertical axis represents the percentage of cell survival of 293T/ACE2 cells after administration of different concentrations of salvianolic acid B compared to 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 only and is not intended to be limiting of the invention.
The following examples evaluate the anti-SARS-CoV-2 infection activity of salvianolic acid B mainly by constructing SARS-CoV-2 live virus and SARS-CoV-2S pseudovirus in vitro cell infection models, and confirm that salvianolic acid B has the ability to resist SARS-CoV-2 infection and simultaneously has the process of inhibiting SARS-CoV-2 from entering target cells, and the inhibition of SARS-CoV-2 from entering target cells by salvianolic acid B inhibits SARS-CoV-2S protein S2 subunit conformational change, thereby inhibiting SARS-CoV-2S protein mediated virus-cell fusion. Provides an application of salvianolic acid B in preparing anti-SARS-CoV-2 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 salvianolic acid B 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 luciferase assay kit adopted in the embodiment of the invention is purchased from Promega corporation of America and comprises luciferase substrate and cell lysate.
The Takara MiniBEST Viral RNA/DNA Extraction Kit, Takara PrimeScript RT reagent Kit with the use of the present invention gDNA Eraser、Takara TBPremix Ex TaqTMIITliRNaseH Plus was purchased from Takara.
Pharmacological experiment part
Example 1 detection of inhibitory Activity of Salvianolic acid B on SARS-CoV-2 in vitro
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. The initial concentration of salvianolic acid B is set to be 200 mu M (solvent is DMSO), the drug is diluted by three times, 3 multiple wells are arranged for each concentration of drug, 7 drug gradients (200, 66.67, 22.22, 7.41, 2.47, 0.82 and 0.27 mu M) are arranged, the solvent dimethyl sulfoxide (DMSO) is set as a control group, the control group is diluted by DMEM culture medium containing 2 percent of fetal calf serum in total volume, and the drug is given with dimethyl sulfoxide in the same volume. 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. Mu.l of the medium containing the drug at the corresponding concentration was added to each well again, and 150. mu.l of the cell culture supernatant was collected after further incubation 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) 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 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 TaqTMII (TliRNaseH Plus, Code No. RR820A) (Standard Curve method: using RBD plasmid of known copy number as standard, the specific primer targets RBD). The reaction solution was prepared on ice as follows:
reagent
Amount used (ul)
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)
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 activity of the salvianolic acid B (Sal-B) against SARS-CoV-25055.47 μ M.
Example 2 detection of inhibitory Activity of Salvianolic acid B on SARS-CoV-2-S protein pseudovirus entry:
1. the method comprises the following steps:
1) SARS-CoV-2-S 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. Changing fresh culture medium 1 hr before transfection, preparing plasmid diluent and transfection reagent (Polyjet) diluent respectively with 100 μ l blank DMEM culture medium at the following ratio (plasmid DNA is extracted by removing endotoxin)Extraction of the kit):
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-micron 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. Cultured in a cell culture chamber at 37 ℃ for 24 hours.
The initial concentration of salvianolic acid B was set at 20 μ M, and 8 concentration gradients (20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 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 a 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 ℃.
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 isovoluminous diluted luciferase substrate according to the specification of a monoluciferase detection kit, immediately carrying out enzyme label instrument detection fluorescence value, and judging the activity of danshensu 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 salvianolic acid B50。
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 B (Sal-B) for inhibiting SARS-CoV-2S protein pseudovirus from entering target cells50It was 0.416. mu.M.
Example 3 Salvianolic acid B inhibits the formation of six helices (6-HB) by SARS-CoV-2HR1P, HR 2P: interfering with the alpha-helical conformation.
During SARS-CoV-2 infection, HR1 and HR2 of SARS-CoV-2S protein S2 subunit can spontaneously polymerize to form hexahelix (6-HB) structure with alpha-helix conformation, thereby promoting membrane fusion of virus and target cell and further promoting virus to enter cell. We synthesized HR1P and HR2P polypeptides located in the core region of 6-HB in vitro and observed whether salvianolic acid B acted on 6-HB.
SARS-CoV-2HR1P：ANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQ (SEQ ID NO：3)
SARS-CoV-2-HR2P：DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL(SEQ ID NO： 4)
Circular Dichroism (CD) uses the difference of absorbance of left and right Circular polarized light by asymmetric molecules to analyze protein secondary conformation (alpha-helix, beta-sheet or random coil), the CD spectrum value of alpha-helix has two negative peaks at 208nm and 222nm, 100% alpha-helix shows-33000 absorption peak at 222nm of CD spectrum, so the proportion of alpha-helix structure in one protein molecule can be determined by X alpha (- [ theta ] theta ═]222) V 33000 calculation.
1. The method comprises the following steps:
1) sample preparation:
HR1P and HR2P polypeptides were prepared as 10 μ M working solution in 50mM PBS, pH 7.2.
System configuration: mu.l of 5mM salvianolic acid B standard concentration 0.15. mu.l were added to 150. mu.l HR1P (10. mu.M), mixed well, and the control group was mixed with 150. mu.l HR1P (10. mu.M) and 0.15. mu.l DMSO, and allowed to react at room temperature for 30 minutes, and 150. mu.l HR2P (10. mu.M) was added and incubation was continued at room temperature for 30 minutes.
2) Round two spectrometer parameter settings (static map):
the detection temperature is 4 ℃, the scanning wavelength range is 190-260nm, the bandwidth is 2nm, the stepping is 1nm, and the time per point is 0.5 second.
3) Sample detection:
adding the sample into a cuvette with 1mm optical path, scanning CD wavelength, and storing CD signal [ theta ]]Value, plot CD curve, and according to [ theta ]]222Values calculate percent alpha-helix.
2. As a result: as shown in fig. 3;
SARS-CoV-2HR1P forms an alpha helix in combination with HR2P, and has a CD spectrum with two negative peaks at 208nm and 222nm (ordinate). The 10. mu.M of HR1P bound α -helices to HR2P at 60.67%, and after 5. mu.M of salvianolic acid B (Sal-B) the percentage of alpha-helices bound to HR1P and HR2P decreased to 22.24%.
Example 4 cytotoxicity assay for Salvianolic acid B
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:
8 concentration gradients were diluted 2-fold prior to dosing in DMEM medium containing 2% total volume of fetal bovine serum.
Vero-E6 cells: the initial concentration was set at 200. mu.M (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625. mu.M), and 100. mu.L of the diluted drug per well was added to Vero-E6 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.
293T-ACE2 cells: the initial concentration was set at 100. mu.M (100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125. mu.M), and 100. mu.L of the diluted drug was added to 1) in 96-well plates, 293T-ACE2 cells, 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.
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. 4 and 5;
salvianolic acid B (Sal-B) has no obvious toxic effect on Vero-E6 cells in 200 μ M and effective concentration range. Salvianolic acid B (Sal-B) had no significant toxic effect on 293T/ACE2 cells (FIG. 5) at 100 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 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
Wuhan Institute of Virology, Chinese Academy of Sciences
Application of <120> salvianolic acid B or pharmaceutically acceptable salt thereof in preparation of anti-SARS-CoV-2 medicines
<130> CP120010256C
<160> 4
<170> PatentIn version 3.3
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<213> Artificial sequence
<400> 2
ctcaagtgtc tgtggatcac g 21
<210> 3
<211> 42
<212> PRT
<213> Artificial sequence
<400> 3
Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser
1 5 10 15
Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn
20 25 30
Ala Gln Ala Leu Asn Thr Leu Val Lys Gln
35 40
<210> 4
<211> 36
<212> PRT
<213> Artificial sequence
<400> 4
Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile
1 5 10 15
Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp
20 25 30
Leu Gln Glu Leu
35