Application of diosmetin or pharmaceutically acceptable salt thereof in preparation of medicine for preventing or treating coronavirus infection diseases

Abstract

The invention provides application of diosmetin or pharmaceutically acceptable salt thereof in preparation of a medicine for preventing or treating coronavirus infection diseases, and relates to the technical field of biological medicines. It is found that diosmetin can be used as a BET inhibitor and a protein degradation agent to effectively exert the anti-coronavirus effect, the natural product diosmetin interacting with BRD4 is screened through a trace thermophoresis technology and a molecular docking technology, and the anti-coronavirus activity of the diosmetin is verified through cell experiments and in-vitro experiments; and the diosmetin has the same in-vitro antiviral activity as the SARS-COV-23CL inhibitor nemategravir, which reveals that the diosmetin has the anti-coronavirus activity and small toxic and side effects, and has the potential of being applied to the preparation of drugs for preventing or treating coronavirus infection diseases.

Description

Technical Field

[0001] The invention provides application of dioscorea or a pharmaceutically acceptable salt thereof in preparing a medicine for preventing or treating a coronavirus infection disease, and relates to the technical field of biomedicine. Background Art

[0002] The widespread prevalence and continuous mutation of coronaviruses pose a huge threat to human health, so researching and developing universal drugs against coronavirus diseases will be an urgent and crucial task.

[0003] Bromodomain (BRDS) is a conserved protein domain that can specifically recognize acetylated lysine residues in proteins. The BET family bromodomain protein BRD4 contains acetylated lysine residues that can bind to histones and other proteins, and plays an important role in regulating genes and controlling cell growth. Many human diseases are closely related to BRD4 protein, such as tumors, autoimmune diseases, and viral infections. Studies have shown that inhibiting BRD4 can loosen the chromatin structure and cause DNA damage, thereby triggering DDR, which in turn activates the cGAS / STING / TBK1 / IRF3 pathway and ultimately activates antiviral innate immunity. This mechanism not only inhibits viral replication, but also promotes the production of antiviral cytokines and enhances the body's antiviral ability. Currently, BET inhibitors are mainly used in cancer treatment, and there are relatively few BET inhibitors used to treat viral infections. Therefore, it is of great significance to study and develop BET inhibitors and drugs that are precisely used to treat coronavirus infections.

[0004] Natural products are a class of substances derived from plants, animals or microorganisms, with unique biological activities and broad application prospects. Natural products have unique value in drug research and development, and their structure and biological activity can serve as the basis for the optimization and design of drug molecules. Diosmetin is a natural flavonoid compound, mainly derived from lemon, chrysanthemum, olive and other plants. Diosmetin has multiple biological activities such as antioxidant, anti-inflammatory, anti-tumor and antibacterial, and is widely used in the prevention and treatment of cancer. Discovering new biological activities and new applications of diosmetin has important practical significance. Summary of the invention

[0005] In order to further meet the needs of developing drugs for preventing or treating coronavirus infections and expand new uses of natural products, the present invention provides the use of dioscorea or a pharmaceutically acceptable salt thereof in the preparation of drugs for preventing or treating coronavirus infections. The natural product dioscorea can be used as an effective protein degrader or an inhibitor of BRD4 protein in the BET family, and is used to prepare drugs for preventing or treating coronavirus infections.

[0006] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0007] The present invention provides an application of diosmetin or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating a coronavirus infection disease. The diosmetin is used to promote the body to produce antiviral factors, reduce virus attachment, degrade viral proteins, and has anti-coronavirus biological activity. The structural formula of the diosmetin is shown in formula (I):

[0008]

[0009] Furthermore, the coronavirus is any one of the coronavirus genera α, β and γ.

[0010] Furthermore, the coronavirus is SARS-CoV-2 of the β genus.

[0011] Furthermore, the IC value of dioscorea in the drug against the novel coronavirus SARS-COV-2 live virus is 50 The value is 11.46μM.

[0012] Furthermore, the effective concentration of diosmetin in the medicine is 1.25 μM to 40 μM.

[0013] Furthermore, the drug contains dioscorea or a pharmaceutically acceptable salt thereof as the only active ingredient.

[0014] Furthermore, the drug also includes a pharmaceutically acceptable carrier and / or excipient.

[0015] Furthermore, the medicine is an oral preparation, an injection or an external preparation.

[0016] Furthermore, the oral preparation is any one of tablets, capsules, pills, powders, granules and syrups.

[0017] Furthermore, the injection is a solution or an emulsion.

[0018] Furthermore, the external preparation is any one of a spray, an aerosol, a gel and a cream.

[0019] The present invention provides an application of diosmetin or a pharmaceutically acceptable salt thereof in the preparation of a BRD4 protein inhibitor. The function of the BRD4 protein inhibitor is to activate an organism to produce antiviral factors.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention proposes that diosmetin can be used as a BRD4 protein inhibitor and protein degrader in the BET family to effectively inhibit the activity of coronavirus, and diosmetin has an anti-novel coronavirus SARS-COV-2 live virus (IC 50 The value is 11.46μM) and Pfizer's currently marketed SARS-COV-23CL inhibitor "specific drug" namatevir (IC 50 The in vitro antiviral activity of diosmetin is comparable to that of 1.5-2.0 μM, and diosmetin has little cytotoxicity and side effects, can significantly reverse the decline in mouse body weight and body temperature, improve the state of mice, and significantly reduce the virus titer in the lungs of mice. Diosmetin or its pharmaceutically acceptable salt can be used to prepare drugs for preventing or treating coronavirus infection diseases; the diosmetin proposed by the present invention can be used to prevent and treat diseases related to the BRD4 protein pathway; the present invention provides a new drug for anti-coronavirus disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are test diagrams for the binding ability of lignin to BRD4 protein; Figure A: MST interaction curve of lignin and BRD4-BD1, Figure B: MST interaction curve of lignin and BRD4-BD2.

[0023] Figure 2 This is a molecular simulation docking diagram of dioscorea lignans and BRD4-BD1 (PDB ID: 3MXF); Figure A: The protein is displayed in Cartoon and line mode, and Figure B: The protein is displayed in Surface mode.

[0024] Figure 3 This is the molecular simulation docking diagram of dioscorea lignans and BRD4-BD2 (PDB ID: 6ZB1); Figure A: The protein is displayed in Cartoon and line mode, Figure B: The protein is displayed in Surface mode.

[0025] Figure 4 The concentration-survival rate curves of RVX-208 and dioscorea lignans on A549-hACE2 cells (Figure A) and Vero-E6 cells (Figure B).

[0026] Figure 5 Figure 1 is a plaque test diagram for inhibiting the activity of the new coronavirus; Figures A to E are the effects of dimethyl sulfoxide, quercetin, kaempferol, luteolin, and geraniol on the reduction of new coronavirus plaques. The first two rows in each attached figure are parallel tests of the low-dose group, and the last two rows are parallel tests of the high-dose group; Figure F is a statistical chart of the reduction of new coronavirus plaques in groups A to E.

[0027] Figure 6 Dioscorea linalool inhibits the IC of the new coronavirus 50Value test graph (Figure A), fitting curve graph of the inhibition results of geraniol on the new coronavirus (Figure B); the first two rows in Figure A are parallel experiments of the namatevir treatment group, the third and fourth rows are parallel experiments of the DMSO treatment group, and the fifth and sixth rows are parallel experiments of the geraniol treatment group.

[0028] Figure 7 This figure shows the effect of cGAS and STING knockout on the anti-COVID-19 function of geraniol.

[0029] Figure 8 This is a diagram showing the effect of geraniol on the expression of SARS-CoV-2 nucleocapsid protein (SARS-CoV-2NP), p-Histone H2A.X, p-STING, IRF3 and p-IRF3 proteins; from top to bottom, they are the protein expressions of SARS-CoV-2 nucleocapsid protein (SARS-CoV-2NP), p-Histone H2A.X, p-STING, IRF3, p-IRF3 and internal reference respectively; the first 6 lanes from left to right are the non-virus infection group, and the last 6 lanes are the coronavirus infection group, among which lanes 1 and 7 are treated with low dose of DMSO, lanes 2 and 8 are treated with high dose of DMSO, lanes 3 and 9 are treated with low dose of RVX-208, lanes 4 and 10 are treated with high dose of RVX-208, lanes 5 and 11 are treated with low dose of geraniol, and lanes 6 and 12 are treated with high dose of geraniol.

[0030] Fig. 9 This is a diagram showing the effect of geraniol on the attachment of the SARS-CoV-2 pseudo-lentivirus; Figures A, B, and C show the attachment of the SARS-CoV-2 pseudo-lentivirus without treatment, after adding DMSO, and after adding geraniol, respectively; the second row of pictures in Figures A, B, and C are enlarged images of the white boxes in the first row of pictures.

[0031] Fig.10 Schematic diagram of drug administration and virus infection in mice.

[0032] Fig.11 The following are the results of in vivo experiments on the effect of geraniol against the new coronavirus; Figure A shows the titer of the new coronavirus in the lungs of mice, Figure B shows the weight change curve of mice; Figure C shows the body temperature change curve of mice. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0034] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0035] The widespread prevalence and continuous mutation of coronaviruses pose a huge threat to human health, so a new, universal anti-coronavirus drug is urgently needed. The BRD4 protein of the BET family bromodomain contains acetylated lysine residues that can bind to histones and other proteins, and plays an important role in regulating genes and controlling cell growth. Studies have shown that inhibiting the BRD4 protein in the BET family can enhance the body's antiviral ability, so there is an urgent need to find a BRD4 inhibitor with low toxicity, high efficiency and high selectivity for the prevention and treatment of coronavirus infection.

[0036] The present invention provides a use of a natural product, diosmetin, or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating a coronavirus infection disease. First, through microthermophoresis technology and molecular docking, it is found that diosmetin can interact with the BRD4 protein of the BET family. Cell experiments and in vivo experiments are used to find that diosmetin can be used as a BET inhibitor and protein degrader. By inhibiting BRD4, DDR reactions are induced, and cGAS-STING pathways are activated, thereby prompting the body to produce antiviral cytokines, reducing virus attachment, and exerting an anti-coronavirus effect. At the same time, diosmetin can exert an anti-coronavirus effect through the BRD4\ACE2 signaling pathway and the NF-κB / PD-1 signaling pathway. Importantly, diosmetin exhibits excellent viral activity (IC 50 The value is 11.46μM), which is comparable to Pfizer's currently marketed SARS-COV-2 3CL inhibitor "specific drug" namatevir (IC 50 The in vitro antiviral activity of this drug is almost of the same order of magnitude, and its cytotoxicity is relatively small, so it has the potential to be used alone or in combination with other drugs to fight viral infections.

[0037] Example 1: Test of the binding ability of dioscorea lignan to BRD4 protein

[0038] 1. Microscale Thermophoresis (MST)

[0039] Microscale Thermophoresis (MST) is an advanced biophysical technology used to measure molecular interactions (such as proteins and small molecule compounds). Microthermophoresis refers to the movement of molecules under a microscopic temperature gradient, and its swimming rate is extremely sensitive to changes in molecular size, charge and solvation entropy at the molecule-solvent interface. Microthermophoresis technology uses infrared laser heating to form a trace temperature gradient field in the capillary, and monitors the changes in the mobility and fluorescence yield of fluorescent labeled molecules in the temperature field to achieve quantitative determination of interactions between biomolecules. MST technology is highly sensitive and can detect tiny changes in molecular structure or conformation. It has high flexibility, is not limited by the molecular weight and type of the sample, has low requirements for sample buffer, and has accurate fluorescence monitoring capabilities.

[0040] The specific experimental steps are:

[0041] (1) A batch of BRD4-BD1 and BRD4-BD2 proteins without His-tag removal were purified and replaced with MST Assaybuffer buffer system using ultrafiltration concentration tubes. The flavonoid compounds to be tested were also diluted to different concentrations using MST Assaybuffer.

[0042] (2) Incubate 10 μL of 300 μM JQ1-CY5 with 90 μL of 10 μM His-tagged target proteins (BRD4-BD1 and BRD4-BD2) in the dark for 30 min for fluorescent labeling, and then use a chromatography column to separate and purify the successfully labeled proteins.

[0043] (3) Use MST Assay buffer to dilute the target protein to a test concentration between 50 nM and 200 nM.

[0044] (4) Mix target protein solutions of different concentrations and test compound solutions of different concentrations evenly, incubate at 4°C for 20 min, and then aspirate the mixed reaction solution using an ordinary capillary.

[0045] (5) MSTpower, set to medium 50% to 70%, tested on the computer, and each set of data was tested twice independently.

[0046] (6) The results were analyzed using NTAnalysis software (NanoTemper Technologies), which automatically calculated the Kd value.

[0047] The results are as follows Figure 1As shown, the affinity activity of diosmetin to the two subtypes (BD1 and BD2) of BRD4 protein is relatively ideal. The Kd values ​​of diosmetin to BD1 and BD2 are 1.46μM and 0.217μM, respectively, and it has a certain selectivity for BD2. Therefore, the present invention decided to test its anti-new coronavirus ability based on diosmetin.

[0048] 2. GLIDE module molecular docking

[0049] First, download the PDB file, optimize the protein and output the glide grid file. Secondly, optimize the compound molecule and output multiple advantageous molecular conformations of the structure. Finally, perform molecular simulation docking calculation and set the docking accuracy to ExtraPrecision to improve the calculation accuracy.

[0050] Molecular simulation docking diagram of geraniol (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and BRD4-BD1 (PDB ID: 3MXF) is shown in the figure Figure 2 As shown in the figure, the carbon group of diosmin and the key N140 amino acid residue of phenolic hydroxyl group have a relatively good combination. Molecular simulation docking diagram of diosmin and BRD4-BD2 (PDB ID: 6ZB1) is shown in the figure. Figure 3 As shown, the carbonyl and phenolic hydroxyl key N140 amino acid residues have a relatively good binding; affected by the difference in amino acid residues between BD2 and BD1, especially the difference between the BD2 protrusion H437 and the BD1 flat D144 in the BCloop region, the benzene ring of geraniol is deeper into the ZAchannel region when binding to BRD4-BD2, and the overall binding is more firmly than BRD4-BD1.

[0051] Example 2: Antiviral activity test of dioscorea in vitro

[0052] 1. Analysis of cell survival rate by dioscorea lignin

[0053] RVX-208 is an effective BET bromodomain protein inhibitor. The MTS method was used to detect the effects of different concentrations of RVX-208 or dioscorea linalool on the viability of Vero-E6 cells and A549-hACE2 cells, and GraphPadprism9.5.1 software was used to fit the drug concentration-cell survival rate curve.

[0054] like Figure 4As shown in the figure, RVX-208 has different degrees of inhibitory effect on the metabolic activity of A549-hACE2 cells. With the continuous increase of the drug concentration, the survival rate of the cells gradually decreased, but the effect of dianthin on A549-hACE2 cells was relatively mild. The effects of RVX-208 and dianthin on the metabolic activity of Vero-E6 cells were relatively mild. According to the changes in the survival rate after the cells were treated with different concentrations of drugs, the IC value of RVX208 on A549-hACE2 cells can be calculated. 50 The value is 25.61 μM, and the IC value for Vero-E6 cells is 50 The value is 210.8 μM; while the IC value of dioscorea for A549-hACE2 cells is 50 The value is 186.8 μM, and the IC value for Vero-E6 cells is 50 The value is 238.2μM.

[0055] 2. Detection experiment of the ability of linalool to resist the new coronavirus

[0056] Drugs against COVID-19 IC 50 Value determination steps:

[0057] (1) Cell division: Using the VTA cell line, dilute the cell suspension to a concentration of 4×10 4 / mL, fill each well of a 24-well plate with 500 μL of the cell suspension, and then place the 24-well plate in a cell culture incubator at 37° C. and 5% CO 2 for three days.

[0058] (2) Gradient dilution of drugs: Set the highest drug concentration to 120 μM, then perform two-fold gradient dilution, set a total of six gradients, and use 5% FBS-DMEM medium for dilution. Aspirate the old medium and add the newly prepared drug-containing medium, 200 μL per well, so that the final drug concentration is 1.25 μM to 40 μM.

[0059] (3) Infect cells: Add 100 μL of diluted virus to each well (the virus titer after dilution is 300 PFU / mL), gently tap the 24-well plate, mark it, and incubate it in a cell culture incubator for 1 hour. Then add 300 μL of 1% methylcellulose solution to each well to stabilize the virus, limit virus spread, and provide a supporting matrix, and incubate it in a cell culture incubator for 72 hours.

[0060] (4) Fixation: Add 10% formalin neutral buffer and incubate at room temperature for 30 minutes to fix the cells and kill the virus. After incubation, rinse off the residual formalin buffer with running water.

[0061] (5) Staining: Drain the water in the wells and add an appropriate amount of 0.9% crystal violet solution to just cover the bottom surface. After staining for 5 minutes, wash off the staining solution with running water and place in a fume hood to dry.

[0062] (6) After drying, take photos, count virus plaques using Mage J software, and then use GraphPad Prism 9.5.1 software to fit the curve and calculate IC 50 value.

[0063] Experimental steps for packaging novel coronavirus pseudotyped lentivirus:

[0064] (1) First, 293T cells were cultured in D10 growth medium until they reached a confluence of 50% to 70% on the second day. For example, a six-well plate contained 5 × 10 5 HEK293T cells.

[0065] (2) Transfection of cells: BioT was used for transfection according to the manufacturer's instructions, and a plasmid mixture was added. The plasmids were all from the SARS-CoV-2 Pseudotype Lentivirus Packaging Kit V2 (purchased from TaKaRa). Taking a six-well plate as an example, 1 μg of Luciferase-IRES-ZsGreen backbone plasmid, 0.22 μg of HDM-Hgpm2, pRC-CMV-Rev1b and HDM-tat1b auxiliary plasmids, and 0.34 g of viral invasion protein S GlycoproteinΔCter were added to each well.

[0066] (3) 24 h after transfection, the culture medium was replaced with fresh D10 growth medium.

[0067] (4) 60 h after transfection, collect the culture medium in each well, centrifuge at 500 g for 5 min, aspirate the supernatant, filter with a 0.45 μm filter membrane, aliquot, and freeze at -80°C.

[0068] Steps for determining the dosage of SARS-CoV-2 pseudotyped lentivirus:

[0069] (1) First, 2 μL of polylysine was added to each well of a 96-well cell culture plate (polylysine can improve cell adhesion and prevent cell damage during infection). After incubation at room temperature for 2 h, the coating solution was discarded and 1.25×10 cells were inoculated in the coated plate using D10 growth medium. 4 HEK293T-hACE2 cells.

[0070] (2) On the second day, first count the number of cells in at least two wells to determine the number of cells at the time of infection.

[0071] (3) Preparation of virus dilutions: Starting from undiluted virus, the virus was diluted three times in a 1:3 gradient, and the final volume in each well was 150 μL.

[0072] (4) Gently aspirate the culture medium on the cells and slowly add the virus dilution solution.

[0073] (5) Adding 3 μL of polyvinyl bromide to each well helps to reduce the charge repulsion between the virus and cells and thus promotes pseudovirus infection.

[0074] (6) At 48 h after infection, luciferase assay was performed using Bright-Glo Luciferase Assay System reagent.

[0075] (7) Remove 100 μL of culture medium from each well, add 30 μL of luciferin reagent, mix thoroughly, transfer all the liquid to a 96-well black bottom plate, incubate at room temperature for 2 h in the dark, and then measure the luminescence intensity using a microplate detector.

[0076] (8) Draw a curve of luminescence intensity and virus dilution, and select the amount of virus that shows a linear relationship between the amount of virus added and the luminescence intensity, and the signal difference between the two is >1000 times as the amount of virus used in subsequent experiments. The final amount of virus used in this experiment was 1:20.

[0077] In order to explore the ability of dianthin to resist the new coronavirus, the commonly used flavonoid compounds quercetin, kaempferol, and luteolin were selected together with dianthin to conduct a virus plaque test (quercetin, kaempferol, and luteolin were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.). Specifically, 2μM and 20μM dianthin, 2.5μM and 40μM quercetin, kaempferol, and luteolin were used to pretreat A549-hACE2 cells for 72 hours, respectively, and dimethyl sulfoxide (DMSO) was set as the control group. After adding virus-infected cells for 24 hours, the culture medium was collected and the virus plaque test was performed (the experimental steps were the same as those of the drug anti-new coronavirus IC 50 value determination step).

[0078] The experimental results are as follows Figure 5 As shown, it can be clearly observed from the virus plaque test plate that in the groups pretreated with dioscorea and quercetin, the high-dose group can clearly see a significant reduction in virus plaques compared with the DMSO negative control group, which strongly proves the activity of the natural small molecule compound dioscorea in fighting the new coronavirus. The other two groups of flavonoids do not have this ability to fight the new coronavirus, which to some extent reflects that this ability is not caused by the broad-spectrum anti-inflammatory and antiviral properties of flavonoids.

[0079] The "special medicine" Nirmatrevir is a SARS-COV-23CL inhibitor launched by Pfizer. In order to further explore the anti-new coronavirus ability of dioscorea, the Nirmatrevir group was set as a positive control and the DMSO group was set as a negative control to conduct a virus plaque test (the experimental steps are the same as those of the drug anti-new coronavirus IC 50 value determination step).

[0080] The experimental results are as follows Figure 6 As shown, from Figure 6 A plaque test plate shows that compared with the DMSO group, the virus plaques in the geranyl group were significantly reduced. Figure 6 From the fitting curve B, it can be seen that dioscorea lignin inhibits the IC of the new coronavirus 50 The value is 11.46 μM, which is almost the same order of magnitude as 3.049 μM in the positive drug namatevir group. As a natural product, its antiviral ability is worthy of recognition.

[0081] 3. The antiviral effect of dianthin is related to the cCAS-STING pathway regulated by BRD4

[0082] A549-hACE2 SicGAS and HEK293T-hACE2 cells pretreated with DMSO and 20 μM dioscorea were infected with SARS-CoV-2 for 72 h, and the culture medium was collected to perform a novel coronavirus plaque assay to determine the virus titer (the experimental steps were the same as those of the drug anti-novel coronavirus IC in Example 2). 50 value determination step).

[0083] from Figure 7 It can be clearly concluded that when cGAS and STING were knocked out, the in vivo antiviral effect of dioscorea linalool disappeared, indicating that the antiviral effect of dioscorea linalool is related to the cCAS-STING pathway regulated by BRD4. At the same time, compared with the infection of normal A549-hACE2 cells with the new coronavirus and the virus titer, it was found that the cells after knocking out cGAS and STING were more susceptible to infection, and the virus titer in the culture medium was higher, reflecting that the cGAS-STING pathway plays an important role in the function of resisting the new coronavirus infection.

[0084] 4. Western blotting to detect the effect of dianthin on the expression of SARS-CoV-2 protein

[0085] Protein sample collection and western blotting experimental steps:

[0086] (1) Protein quantification: This experiment uses the DC proteinAssay Kit II (purchased from BioRad) for protein quantification, and the standard protein is bovine serum albumin (BSA). First, add the prepared BSA standard protein (1g / mL) to the microplate at a volume of 0μL, 1μL, 2μL, 3μL, 4μL, 5μL, 8μL, and 10μL, and set up two replicate wells. Pipette 1μL of the protein to be tested and add it to the microplate, and also set up two replicate wells. Add 20μL of reagent S to 1mL of reagent A, mix well, and use a dispenser to add 25μL to each well of the microplate. Then use a dispenser to add reagent B to the microplate at a volume of 200μL per well, taking care not to generate bubbles. Place the microplate on a microplate oscillator and incubate for 5 minutes. Then use a microplate detector to read the absorbance value at 750nm. Then, the standard curve equation is fitted with the BSA concentration and the absorbance value, and the concentration of the protein to be tested is calculated using the standard curve equation and the absorbance value of the protein to be tested.

[0087] (2) Sample preparation: First, add equal amounts of protein to eight tubes in sequence, then add 20×DTT and 4×NuPAGE LDS sample buffer, and then fill up to the same volume with double distilled water. Heat at 75°C for 10 min to destroy the secondary and tertiary structures of the protein and convert the protein into linear, negatively charged polypeptides in preparation for subsequent SDS-PAGE gel electrophoresis.

[0088] (3) SDS-PAGE gel electrophoresis: The electrophoresis condition was set at 60 V for 30 min and then changed to 90 V for 75 min.

[0089] (4) Transfer: This experiment uses the wet transfer method. Place the transfer splint and set up the transfer "sandwich" in the order of transfer plate (negative electrode), cotton pad, filter paper, SDS-PAGE gel, nanofiber membrane, filter paper, cotton pad, and transfer plate (positive electrode). Note that the cotton pad, filter paper, and nanofiber membrane should be moistened with transfer solution in advance. Use a roller to gently roll between each layer to remove bubbles. Then put the "sandwich" into the electrophoresis tank filled with transfer solution, add an ice box, and transfer overnight at 25V.

[0090] (5) Blocking: Use 6 mL of 5% (w / v) skim milk per membrane and shake gently on a seesaw shaker at room temperature. Block for 30 min to block nonspecific binding sites. After blocking, wash with PBST 4 times, 5 min each time. Mark the area without protein sample and protein marker.

[0091] (6) Primary antibody incubation: Prepare an appropriately diluted primary antibody, add it to the container where the membrane is located, and incubate at room temperature for 4 hours, or at 4°C overnight. After the primary antibody incubation is completed, wash with PBST 4 times, 5 minutes each time, to remove unbound primary antibody.

[0092] (7) Secondary antibody incubation: Prepare a properly diluted secondary antibody and add it to the container where the membrane is. Incubate at room temperature for 1 hour. Wash as before. After washing, place the membrane in PBS.

[0093] (8) Exposure: Mix solution A and solution B of the color developing solution in a ratio of 1:1, add the color developing solution to allow the color developing solution to evenly adhere to the membrane surface, then use an exposure device to expose, save the image, and analyze it according to the loading order.

[0094] The above protein sample collection and protein immunoblotting experimental steps were used to determine the effect of dianthin on the expression of SARS-CoV-2 protein. The experimental results are shown in Figure 8 In the high-dose group of diosmin, a significant decrease in the expression of the new coronavirus nucleocapsid protein (SARS-CoV-2NP) was observed, which is consistent with the plaque test results of diosmin against the new coronavirus. The expression of phosphorylated STING protein, phosphorylated IRF3, and P-Histone H2A.X protein also increased significantly in the group with diosmin, which is consistent with the antiviral mechanism of diosmin after inhibiting BRD4.

[0095] 5. Dioscorea lignin reduces virus attachment experiment

[0096] Confocal microscopy was used to observe the attachment of SARS-CoV-2 pseudotyped lentivirus (PSC2) infected with DMSO and treated with dioscorea. Since PSC2 contains GFP protein, it emits green fluorescence, and since the cell membrane is stained with CellMaskDeep red dye, the cell membrane appears red. Fig. 9 It can be clearly seen that in the group using dioscorea lignans, the green fluorescence was greatly reduced, indicating that dioscorea lignans did reduce the attachment of viruses.

[0097] Example 3: In vivo experiment of dioscorea lignin against novel coronavirus

[0098] Fig.10 Schematic diagram of drug administration and virus infection in B6 mice.

[0099] The animal experimental samples in this example are healthy C57BL / 6J mice (hereinafter referred to as B6 mice), purchased from Jackson Laboratory, SPF level. The experimental mice were kept at the Animal Center of the University of Arizona, USA, with a room temperature of 76°F, a humidity of 50%, 12h alternating light, and free access to water and food. All experimental designs were carried out in accordance with the relevant management regulations of the University of Arizona. The surgical instruments were strictly sterilized, and the animals were strictly sterilized after surgery. All experiments related to SARS-CoV-2 were carried out in the Biosafety Level 3 Laboratory of the University of Arizona.

[0100] In order to verify the effect of the present invention, this embodiment uses SARS2-N501YMA30 virus (referred to as MA30 virus) to infect mice, and then uses dioscorea to treat mice infected with MA30 virus. The preparation method of MA30 virus is: using the rSARS2-N501Yp0 virus strain provided by Professor Stanley Perlman's laboratory at the University of Iowa, this virus strain is obtained by inserting a sequence encoding N501Y mutation in the genome of Wuhan-Hu-1 (SARS-CoV-2) strain using reverse genetics technology, and this virus strain has a wide range of applications and recognition in related fields. 50μL rSARS2-N501Yp0 virus strain was inoculated into 8-10 week old BALB / c mice by nasal administration, and the mice were euthanized 3 days later, lung tissue was collected, 1.5mm zirconium oxide ceramic beads were used, PBS was added to grind the tissue, and it was frozen and thawed twice at -80°C, and the supernatant was taken by centrifugation, and the virus was labeled as SARS2-N501YMA1. The SARS2-N501YMA1 coronavirus was used to infect mice, and the virus was passaged to the 30th generation to obtain the SARS2-N501YMA30 virus.

[0101] The specific modeling method is as follows: First, B6 mice were pretreated with 100 mg / kg of dioscorea by intraperitoneal injection, and 10% v / v DMSO + 90% v / v castor oil was set as a negative control group. The weight and temperature of the mice were monitored and the status of the mice was scored every day. On the third day, 5×10 4 PFU of MA30 virus was intranasally administered to mice until the 8th day. The lung tissues of mice were collected and weighed on the 5th day after infection. The virus titer (LD 50 ) were used to treat lung tissue samples, and then the virus titer was detected by virus plaque assay using VTA cells, and then the mouse lung tissue weight was used for correction and a bar graph was drawn. The difference between the two groups was detected by T test.

[0102] The experimental results after administration are shown in Fig.11 .from Fig.11 As can be seen in Figure A, the virus titer of the group treated with dioscorea was significantly reduced. From the weight change curve in Figure B, it can be observed that the weight of mice in the group treated with dioscorea suddenly dropped on the 5th day, but began to gradually recover on the 6th day, while the weight of mice in the negative control group showed a progressive decrease. A similar trend was also observed in the temperature change curves of the two groups of mice in Figure C, which indirectly reflects that the mice treated with dioscorea are in a better state.

[0103] In summary, the present invention comprehensively reveals that the natural product dioscorea lignin, as a BRD4 protein inhibitor and protein degrader of the BET family, can effectively protect mice infected with coronavirus, has significant antiviral efficacy, and has the potential to become a drug for preventing and treating coronavirus infection through cell level experiments and animal experiments. The dioscorea lignin provided by the present invention provides new prevention and control drugs and new ideas for viral infection, enriches the application of natural product resources, and has broad promotion and application value.

[0104] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of dioscorea or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating coronavirus infection diseases.

2. The use of diosmetin or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing or treating coronavirus infection diseases, characterized in that: The coronavirus is any one of the coronavirus genus α, β and γ.

3. The use of diosmetin or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing or treating coronavirus infection diseases, characterized in that: The coronavirus is SARS-CoV-2 of the β genus.

4. The use of dioscorea or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing or treating coronavirus infection diseases, characterized in that: The effective concentration of dioscorea in medicine is 1.25μM~40μM.

5. The use of diosmetin or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing or treating coronavirus infection diseases, characterized in that: The medicine uses dioscorea or its pharmaceutically acceptable salt as the only active ingredient.

6. The use of diosmetin or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing or treating coronavirus infection diseases, characterized in that: The drug includes a pharmaceutically acceptable carrier and / or excipient.

7. The use of diosmetin or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing or treating coronavirus infection diseases, characterized in that: The medicine is any one of an oral preparation, an injection and an external preparation.

8. The use of diosmetin or a pharmaceutically acceptable salt thereof according to claim 7 in the preparation of a drug for preventing or treating coronavirus infection diseases, characterized in that: The oral preparation is any one of tablets, capsules, pills, powders, granules and syrups.

9. The use of diosmetin or a pharmaceutically acceptable salt thereof according to claim 7 in the preparation of a drug for preventing or treating coronavirus infection diseases, characterized in that: The injection is a solution or an emulsion.

10. The use of diosmetin or a pharmaceutically acceptable salt thereof according to claim 7 in the preparation of a drug for preventing or treating coronavirus infection diseases, characterized in that: The external preparation is any one of a spray, an aerosol, a gel and a cream.

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