Anti-coronavirus traditional Chinese medicine composition and mechanism research method thereof

Abstract

The invention discloses an anti-coronavirus traditional Chinese medicine composition and a mechanism research method thereof, belongs to the technical field of biological medicines, and solves the problems that the existing coronavirus is difficult to treat and the treatment mechanism is not clear yet. Comprising 10-20 parts of herba schizonepetae, 10-20 parts of radix saposhnikoviae, 10-20 parts of folia perillae acutae, 10-20 parts of pericarpium citri reticulatae, 10-20 parts of roasted rhizoma atractylodis macrocephalae, 10-20 parts of poria cocos, 10-20 parts of fructus aurantii, 10-20 parts of radix bupleuri, 10-20 parts of radix peucedani and 5-15 parts of honey-fried licorice root. According to the application disclosed by the invention, the PI3K-AKT signal channel is regulated and controlled by interfering the combination of ACE2 and RBD and targeting an AKT1 target spot, so that the expression of SARS-CoV-2 infected host cells and cytokines IL1b, TNF and IL6 is inhibited, and the inflammatory response is alleviated.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-coronavirus Chinese medicine composition and a method for studying its mechanism. Background Art

[0002] Coronavirus is a group of single-stranded positive-sense RNA viruses with an envelope. Currently, there are seven known types that can infect humans: human coronavirus 229E, human coronavirus OC43, severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63, human coronavirus HKU1, Middle East Respiratory Syndrome Coronavirus (MARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which mainly cause respiratory system infections.

[0003] The main clinical symptoms of SARS-CoV-2 infection are fever, cough, and pain, leading to respiratory failure, multiple organ failure, and even death. The severity of the disease is generally related to the patient's immune system status and the presence or absence of underlying diseases such as cardiovascular disease, hypertension, and diabetes. Healthy people can generally recover 2-4 weeks after treatment. The virus is mainly transmitted through contact with infected body fluids, respiratory droplets and aerosols, etc. It can be deposited on the surface of objects and can survive for several days under suitable conditions. Therefore, the virus is highly contagious, and no group that is not easily infected has been found. In addition, due to the continuous mutation of the virus, people who are infected or vaccinated can still be infected again, and even face the risk of breakthrough infection and development of serious diseases.

[0004] At present, the main mechanisms for treating and preventing SARS-CoV-2 are: preventing the virus from entering host cells, inhibiting viral replication, and immune regulation. The RBD region on the surface of the new coronavirus is the key to binding to the ACE2 receptor on the cell membrane to infect cells, so targeting this binding effect is an important direction for the development of drugs that hinder virus entry. For example, antibodies targeting the RBD region or ACE2 receptor, convalescent plasma containing antiviral antibodies, vaccines, ACE2 receptor inhibitors, and Umifenovir that interacts with viral glycoproteins. Another treatment direction is to hinder the replication and release of the virus: targeting RNA-dependent RNA polymerase (RNA-dependent RNA polymerase, RdRp), such as chloroquine (chloroquine), Remdesivir (Remdesivir), etc. to hinder viral RNA synthesis; inhibiting viral protease activity, such as Lopinavir-Ritonavir (Lopinavir-Paxlovid). For patients with excessive immune response, anti-inflammatory treatment is also necessary, such as the inflammatory factor antibody IL-6 antibody tocilizumab, the inflammatory pathway inhibitor JAK inhibitor Ruxolitinib, etc. In addition, the viral 3C-like protein (3C-like protease inhibitor, 3CLpro) is also a promising potential therapeutic target because it is an enzyme necessary for viral replication and is highly conserved, with broad-spectrum resistance to coronaviruses.

[0005] Traditional Chinese medicine (TCM) has a history of more than 2,000 years in treating infectious diseases, and has accumulated more than 10,000 Chinese herbal medicines and 100,000 prescriptions. Compared with other anti-coronavirus drugs, TCM has the characteristics of multiple components, multiple targets, and fewer side effects. It has played a unique advantage in shortening treatment time, relieving symptoms, and reducing the severity of the disease. In addition, TCM has made outstanding contributions to the prevention and control of SARS, and also provided reference value for SARS-CoV-2.

[0006] Therefore, finding drugs that can fight both viral infection and immunity and treating them as early as possible is an important means to prevent severe SARS-CoV-2 and complications. Summary of the invention

[0007] The present invention provides an anti-coronavirus Chinese medicine composition and a method for studying its mechanism, which have solved the technical problems of the difficulty in existing coronavirus treatment and the unclear treatment mechanism.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The present application provides an anti-coronavirus Chinese medicine composition, which is composed of the following components in parts by weight: 10-20 parts of Schizonepeta, 10-20 parts of Saposhnikovia, 10-20 parts of Perilla leaves, 10-20 parts of dried tangerine peel, 10-20 parts of stir-fried Atractylodes, 10-20 parts of Poria, 10-20 parts of Citrus aurantium, 10-20 parts of Bupleurum, 10-20 parts of Peucedanum, and 5-15 parts of roasted Licorice.

[0010] Furthermore, it is composed of the following components in parts by weight: 15 parts of Schizonepeta, 15 parts of Saposhnikovia, 15 parts of Perilla leaves, 15 parts of dried tangerine peel, 15 parts of stir-fried Atractylodes, 15 parts of Poria, 15 parts of Citrus aurantium, 15 parts of Bupleurum, 15 parts of Peucedanum, and 10 parts of roasted Licorice.

[0011] Furthermore, the antiviral and anti-immune compounds in the traditional Chinese medicine composition are: hesperidin, β-sitosterol, naringenin and luteolin-7-O-glucoside.

[0012] Furthermore, the Chinese medicine composition exerts an antiviral effect by inhibiting the binding of S protein to ACE2 and the PI3K-AKT signaling pathway.

[0013] The present invention provides a method for studying the mechanism of an anti-coronavirus Chinese medicine composition, which specifically comprises the following steps:

[0014] S1. Construction of pseudovirus: The original strain and its variant pseudovirus carrying green fluorescent protein GFP were packaged using the HIV-1 three-plasmid system, concentrated using the PEG8000 method, and the infection ability and titer of the pseudovirus were verified by infection experiments;

[0015] S2. Evaluation of the anti-pseudovirus infection effect of the Chinese medicine combination: The cytotoxicity of the compound Chinese medicine was detected by the CCK8 method to determine the safe action time and concentration; through the infection inhibition experiment, two pseudoviruses and three compound drugs were co-cultured separately, and the number of green fluorescent cells was observed under a fluorescence microscope to determine the effect of the compound Chinese medicine on the virus infection activity.

[0016] S3. Exploration of the potential mechanism of Chinese herbal medicine combination against coronavirus: exploring the potential mechanism through network pharmacology and molecular docking technology;

[0017] S4. Real-time fluorescence quantitative PCR technology was used to detect the anti-inflammatory effect of drugs on pseudovirus infection: total cell RNA was extracted using the trizol RNA extraction method, and after reverse transcription, fluorescence quantitative PCR technology was used to detect the effects of the three compounds on the expression of IL1b, IL6, and TNF.

[0018] Furthermore, in step S1, the three-plasmid system includes pCDH-GFP, psPAX2 and the envelope plasmid of the spike protein.

[0019] Furthermore, in step S1, the concentrated virus titer is calculated as follows:

[0020] The titer of the concentrated virus = the number of fluorescent cells in the field of view × dilution factor × 1000 mL / the volume of the original concentrated virus added.

[0021] Furthermore, the step S3 specifically includes the following sub-steps:

[0022] S31. Use TCMSP to determine the target of the compound, and GeneCards, TTD, and OMIM databases to determine the targets related to pneumonia caused by the new coronavirus;

[0023] S32. Use Cytoscape to draw a “key compound-target network diagram” to obtain core drug small molecule compounds;

[0024] S33, use PPI to generate protein interaction network diagram and obtain core targets;

[0025] S34, GO and KEGG enrichment analysis was performed using the DAVID database;

[0026] S35. Use AutoDock to perform molecular docking of key small molecule compounds and screened key targets.

[0027] Beneficial effects achieved by the present invention:

[0028] The anti-coronavirus Chinese medicine composition and the mechanism research method provided by the present invention are based on the SARS-CoV-2 (original strain and Omicron variant) pseudovirus of the HIV-1 three-plasmid system, and the antiviral effect of SCZYY1 and 2 is evaluated using the pseudovirus system. In-depth research has found that the Chinese medicine composition can interfere with the binding of ACE2 and RBD, and target the AKT1 target to regulate the PI3K-AKT signaling pathway, thereby inhibiting SARS-CoV-2 infection of host cells and the expression of cytokines IL1b, TNF, and IL6, and reducing inflammatory responses. In addition, we have also obtained key antiviral and anti-immune compounds. These findings provide clues and basis for the study of traditional Chinese medicine against SARS-CoV-2 infection, and lay a scientific foundation for further drug development and clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the map of the plasmid pCDH-GFP of the present invention;

[0030] Figure 2 is a map of the plasmid psPAX2 of the present invention;

[0031] Figure 3The toxicity test results of the Chinese medicine compound 1 on HEK293T cells;

[0032] Figure 4 The results of the toxicity test of the Chinese medicine compound 2 on HEK293T cells;

[0033] Figure 5 The toxicity test results of Lianhua Qingwen Capsule of the present invention on HEK293T cells;

[0034] Figure 6 It is a Venn diagram of the intersection target of the Chinese medicine compound 1 of the present invention and the virus;

[0035] Figure 7 It is a Venn diagram of the intersection target of the Chinese medicine compound 2 of the present invention and the virus;

[0036] Figure 8 This is a target pathway diagram for the Chinese herbal compound for treating viruses.

[0037] Reference numerals:

[0038] The drawings are only used for illustrative purposes and should not be construed as limitations on this patent. In order to better illustrate this embodiment, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of the actual product. For those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted. The same or similar numbers correspond to the same or similar parts. The terms describing the positional relationship in the drawings are only used for illustrative purposes and should not be construed as limitations on this patent. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0040] The technical solution of the present invention is described in detail below in conjunction with the specific drawings.

[0041] Example 1: Construction of SARS-CoV-2 pseudovirus system

[0042] In this experiment, the plasmid containing the S protein gene of the SARS-CoV-2 Omicron variant was provided by Onogene. The plasmid containing the S protein gene of the original SARS-CoV-2 strain and the VSV-G glycoprotein gene, the transfer plasmid pCDH-GFP carrying the GFP reporter gene, and the lentiviral packaging plasmid psPAX2 were all purchased from Miaoling Biotechnology. The map of the plasmid pCDH-GFP is shown in Figure 1 The map of plasmid psPAX2 is shown in Figure 2 All plasmids were verified by sequencing as indicated.

[0043] HEK293T cells were maintained in our laboratory and cultured in DMEM complete medium.

[0044] 1.1 Preparation of bacterial culture

[0045] (1) Glycerol bacteria recovery: Take the glycerol bacteria and continuously streak them on LB solid culture plates containing resistance to isolate and screen out single clones.

[0046] (2) Plasmid cloning: Pick a single colony and amplify it in small quantities in 5 mL of resistant LB liquid medium, shake it at 37°C, 220 r / min; after shaking culture for 8 h, take 1 mL of the bacterial solution and amplify it in large quantities in 200 mL of LB liquid medium, shake it at 37°C, 220 r / min for 20 h.

[0047] 1.2 Extraction of plasmid

[0048] The plasmid was extracted using a centrifugal column type plasmid extraction kit, and then the concentration and purity were detected using an ND2000 UV spectrophotometer. If A260 / A280 = 1.8-2.0, it indicated that the plasmid purity was good and the extraction was successful.

[0049] 1.3 Pseudovirus construction

[0050] (1) Plant 10 6 HEK293T cells were cultured overnight until the cell density reached about 80% and then transfection could be performed. Before transfection, the supernatant was carefully discarded with a pipette, 2 mL of complete culture medium was added to the cells adhered to the wall, and the cells were placed in the incubator.

[0051] (2) Prepare transfection complex: Add 4 μg of plasmid to 50 μL serum-free DMEM and mix thoroughly to prepare plasmid dilution solution; add 10 μL transfection reagent to 50 μL serum-free DMEM, mix thoroughly, and leave at room temperature for 5 minutes to complete the preparation of transfection agent dilution solution. Mix the transfection agent dilution solution and plasmid dilution solution thoroughly and leave at room temperature for 15 minutes to prepare transfection complex.

[0052] (3) Add the transfection complex to the cells, gently shake the culture medium to mix, and place in an incubator for culture.

[0053] 1.4 Collection and concentration of pseudoviruses

[0054] Collect cell supernatants 24h and 72h after transfection, combine the collected supernatants and centrifuge at 4°C, 4000rpm for 10min to remove cell debris, filter the supernatant with a 0.45μm filter and collect it in a new centrifuge tube, use PEG8000 to concentrate the cells, mix 5×PEG8000 NaCl solution with the virus solution at a ratio of 1:4, mix it upside down every 20min, and do it 5 times in total. Leave it at 4°C overnight, centrifuge it at 4°C, 7000rpm for 20min, discard the supernatant, add an appropriate amount of serum-free DMEM to resuspend it, concentrate the virus 100 times, and store it in aliquots at -80°C.

[0055] 1.5 Pseudovirus titer determination

[0056] (1) Plant 10 4 HEK293T cells were cultured overnight until the cell density reached about 80%;

[0057] (2) Perform gradient dilution of the pseudovirus solution. Take 5 1.5 mL clean centrifuge tubes, number them 0 to 4, add 396 μL DMEM complete medium to tube 0, add 360 μL DMEM complete medium to tubes 1 to 4, add 4 μL of concentrated pseudovirus solution to tube 0, pipette and mix well, take 40 μL to tube 1, mix well, add 100 μL / well to the cells, set up 3 replicates, take 40 μL of the remaining pseudovirus solution to well 2, and so on. Get 4 concentration gradients, three replicates, and set up cell controls. Place in a cell culture incubator and incubate for 24 hours, then replace with fresh DMEM complete medium. After continuing to culture for 36 hours, use a fluorescence microscope to observe the amount of green fluorescence.

[0058] The titer of concentrated virus is calculated as follows:

[0059] Titer of concentrated virus = number of fluorescent cells in the field of view × dilution factor × 1000mL / volume of original concentrated virus added

[0060] 1.6 Experimental Results

[0061] (1) Construction of SARS-CoV-2 (wild type), SARS-CoV-2 (Omicron) and VSV-G pseudoviruses using a lentiviral packaging method. The transfer plasmid pCDH-GFP and the packaging plasmid psPAX2 were co-transfected with envelope plasmids carrying S (wild type), S (Omicron) and VSV-G proteins into HEK293T cells, respectively, to construct three pseudoviruses carrying GFP, which expressed green fluorescent protein after cell infection.

[0062] (2) In order to detect the ability and titer of the constructed pseudovirus to infect host cells, a series of concentration gradients of SARS-CoV-2 (wild type), SARS-CoV-2 (Omicron) and VSV-G pseudoviruses were co-incubated with HEK293T cells, and the number of green fluorescent cells was observed under a fluorescence microscope. All three pseudoviruses have the ability to infect cells and can successfully express GFP. According to the formula calculation, the titer of the concentrated SARS-CoV-2 (wild type), SARS-CoV-2 (Omicron) and VSV-G pseudoviruses reached 10 8 TU / mL, which can meet the experimental needs.

[0063] Example 2: Study on the Antiviral Infection of Compound Chinese Medicine

[0064] Chinese medicine compound 1 (SCZYY1): Schizonepeta 10g, Saposhnikovia 10g, Perilla leaf 10g, Tangerine peel 10g, Stir-fried Atractylodes 10g, Roasted Licorice 5g.

[0065] Chinese medicine compound 2 (SCZYY2): 15g of Schizonepeta, 15g of Saposhnikovia, 15g of Perilla leaf, 15g of dried tangerine peel, 15g of stir-fried Atractylodes, 15g of Poria, 15g of Citrus aurantium, 15g of Bupleurum, 15g of Peucedanum, and 10g of roasted Licorice root.

[0066] The raw materials of the Chinese medicine compound were purchased from the Affiliated Hospital of Chengdu Medical College.

[0067] Lianhua Qingwen Capsule (LH): roasted ephedra, stir-fried bitter almond, forsythia, honeysuckle, rhubarb, houttuynia, patchouli, rhubarb, gypsum, menthol, licorice, isatis root, and rhodiola rosea, purchased from Shijiazhuang Yiling Pharmaceutical.

[0068] 2.1 Screening of safe concentration of compound Chinese medicine

[0069] (1) Solution preparation: First, use dimethyl sulfoxide to dissolve each Chinese medicine to the concentration of the stock solution (500 mg / mL). Then dilute the stock solution to an appropriate concentration using DMEM cell culture medium containing 5% fetal bovine serum as the working solution;

[0070] (2) Cell treatment: 10 cells were seeded in a 96-well cell culture plate. 4 / well density of HEK293 cells, overnight to make the cell density reach about 80% for cytotoxicity experiment. Aspirate the culture medium and add each compound Chinese medicine solution of a series of concentrations, 100μL / well, set up 3 replicates, and set the cells without drugs as blank control group. Place the cells in a cell culture incubator and incubate for 24h and 48h, then use CCK8 method to detect cell activity;

[0071] (3) Detection of cell activity by CCK8 method: Under light-proof conditions, the culture medium was aspirated, and DMEM culture medium containing 10% CCK8 reagent was added to the plate, which was placed in a 37°C cell culture incubator for 1 h. A background control (CCK8 reagent + DMEM culture medium) was set, and the absorbance OD value at a wavelength of 450 nm was detected by an ELISA instrument. The cell survival rate was calculated according to the following formula:

[0072] Cell survival rate = (OD value of drug treatment group - OD value of background control group) / (OD value of blank control group - OD value of background control group).

[0073] 2.2 Effects of compound Chinese medicine on pseudovirus infection

[0074] (1) Cell treatment: 10 cells were seeded in a 96-well cell culture plate. 4 HEK293 cells with a density of 1 / well were added, and the cell density was made to reach about 80% overnight for infection inhibition experiment. The culture medium was aspirated and 100 μL of a mixture of three compound Chinese medicine preparations with a series of concentrations and three pseudoviruses of S-wild type, S-Omicron and VSV-G was added. The amount of pseudovirus was MOI=1. At the same time, a cell control group (cell+culture medium), a virus control group (cell+virus+DMSO+culture medium), and each drug control group (cell+virus+three drugs of different concentrations+culture medium) were set up. After being placed in a cell culture box and incubated for 24 hours, the supernatant was discarded and replaced with DMEM cell culture medium. After continuing to incubate for 36 hours, the pseudovirus infection effect was observed by fluorescence microscopy.

[0075] The pseudovirus infection status (i.e., relative fluorescence expression) was calculated according to the following formula:

[0076] Relative fluorescence expression = number of fluorescent cells in the drug control group / number of fluorescent cells in the virus control group.

[0077] 2.3 Cytotoxicity assay of SCZYY1, SCZYY2 and Lianhua Qingwen capsules The cytotoxicity of the three compound Chinese medicines on HEK293T cells was tested:

[0078] SCZYY1 and 2 were treated with a series of concentration gradients (0.5, 1.5, 2.5, 3.5, 4.5, 5.5, 6.5 mg / mL) and Lianhua Qingwen capsule (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 mg / mL) for 24 and 48 h, respectively. CCK8 solution was used to detect cytotoxicity, and GraphPadPrism was used to calculate the drug concentration at which 50% of the host cells were destroyed (CC 50 ) and the drug concentration at which 90% of host cells are destroyed (CC 90 );SCZYY1 and SCZYY2 acted on HEK293T cells, and CC50 4.0mg / mL, 3.0mg / mL respectively; CC 90 3.1mg / mL and 3.0mg / mL respectively; CC after 48h 50 6.1mg / mL and 6.5mg / mL respectively; CC 90 The CC of Lianhua Qingwen Capsules was 2.0mg / mL and 1.0mg / mL respectively after 24 hours. 50 =1.1mg / mL, CC 90 =0.7mg / mL; CC at 48h 50 =1.5mg / mL, CC 90 =0.8mg / mL. The cytotoxicity test results of the three traditional Chinese medicines are as follows Figure 3-Figure 5 As shown, when the concentration was less than 3.0 mg / mL and 0.7 mg / mL at 24 h, SCZYY1, SCZYY2 and Lianhua Qingwen Capsules had no obvious toxicity to HEK293T cells.

[0079] 2.4 Effects of SCZYY1, SCZYY2 and Lianhua Qingwen Capsules on SARS-CoV-2 (wild-type, Omicron) pseudovirus and VSV pseudovirus:

[0080] In order to detect the effects of SCZYY1, SCZYY2 and Lianhua Qingwen capsules on the infection of new coronavirus (wild type, Omicron) pseudovirus and VSV pseudovirus, an infection inhibition test was used. The compound Chinese medicine solution with a series of concentrations (SCZYY1 and SCZYY2: 1.0, 2.0, 3.0 mg / ml; LH: 0.3, 0.5, 0.7 mg / ml) was incubated with the three pseudoviruses for 24 hours. After 48 hours of culture, the number of green fluorescent cells was observed under a fluorescence microscope. The three prescription Chinese medicines with a series of concentrations showed inhibitory effects on the new coronavirus wild type and Omicron pseudovirus, and the effect was stronger with the increase of drug concentration. GraphPadPrism was used to calculate the half-inhibitory concentration (IC50) of SCZYY1, SCZYY2 and LH on the infection of new coronavirus wild type pseudovirus. 50 ) were 6.615 mg / ml, 2.503 mg / ml and 1.086 mg / ml respectively; selectivity index (SI = CC 50 / IC 50 ) were 0.60, 1.20, and 1.01 respectively; IC 50They were 5.402 mg / ml, 2.879 mg / ml, and 1.825 mg / ml, respectively; the SIs were 0.74, 1.04, and 0.60, respectively. In comparison, SCZYY1 had a poor anti-COVID-19 wild-type pseudovirus infection effect, while SCZYY2 and LH both showed good anti-viral effects, and SCZYY2 had the best anti-viral infection effect. Compared with the COVID-19 wild-type pseudovirus strain, the inhibitory efficiency of the three drugs on the Omicron pseudovirus strain was slightly reduced, and LH showed greater cytotoxicity.

[0081] Example 3: Exploring the mechanism of action of two compound Chinese medicines in treating pneumonia caused by the new coronavirus based on network pharmacology and molecular docking

[0082] (1) Obtaining active ingredients

[0083] The TCMSP database, a data analysis platform for systems pharmacology of traditional Chinese medicine, was used to obtain the corresponding active ingredients and targets of SCZYY1 and SCZYY2. The protein sequence and functional information resource Uniprot database was used to match the related targets of these two compound traditional Chinese medicines with the gene names, and the duplicate values ​​were deleted to obtain the targets.

[0084] (2) Acquisition of disease targets for pneumonia caused by the new coronavirus

[0085] The GeneCards disease database, the drug target database TTD, and the online "Mendelian Inheritance in Man" database OMIM were used to search for disease targets using the keyword "pneumonia caused by the new coronavirus". The collected results were combined to establish genes related to pneumonia caused by the new coronavirus.

[0086] (3) Acquisition of intersection targets

[0087] The targets of the two compound Chinese medicines and the targets of pneumonia caused by the new coronavirus were compared by VENNY, and the Venn diagram of the intersection targets was calculated and drawn, as shown in Figure 6 and Figure 7 As shown, the targets of the two compound Chinese medicines for treating pneumonia caused by the new coronavirus were obtained.

[0088] (4) Construction of the “key active ingredients-disease targets” network diagram

[0089] In order to obtain the key compounds and key targets of SCZYY1 and 2 for the treatment of pneumonia caused by the new coronavirus, we mapped the targets of traditional Chinese medicine for treating diseases to the corresponding small molecule compounds, imported them into Cytoscape for visualization analysis, and performed topological analysis through Network Analyzer to screen out key compounds and key targets, and construct a "key active ingredient-disease target" network diagram.

[0090] (5) PPI network construction

[0091] The intersection targets were imported into the STRING protein interaction database for Protein-Protein Interaction (PPI) network analysis. The species was limited to "Homo Sapiens" and the "Minimum Required Interaction Score" was set to the highest confidence (0.9) to construct the PPI network and produce the PPI protein interaction network diagram.

[0092] (6) GO and KEGG enrichment analysis

[0093] The Metascape database was used to perform KEG and GO enrichment analysis on disease-drug intersection targets, and the species selected was "Homo Sapiens". The drawing of the core pathway diagram was completed based on the KEGG database. GO enrichment analysis mainly included biological process (BP), cellular component (CC) and molecular function (MF). The statistical significance threshold was set to p < 0.01, and the data results were visualized and analyzed using the microbial information platform.

[0094] (7) Molecular docking of active ingredients and core targets

[0095] The active ingredients of the two compound Chinese medicines obtained in (4) were selected as ligands, and ACE2, Spike RBD region, 3CLpro, AKT1, and STAT3 were selected as receptors for molecular docking. The 3D structures of these active ingredients were obtained through the TCMSP database and saved in the "mol 2" format. The 3D structures of these six proteins were downloaded from the RCSB PDB protein database in the "PDB" format. The receptor and ligand formats were converted to the "pdbqt" format using PyMOL 2.4, and the molecular docking was performed using AutoDock Vina 1.1.2 software to calculate the binding energy. PyMOL 2.4 was also used to visualize the binding sites.

[0096] This example adopts the methods of network pharmacology and molecular docking to collect targets related to SCZYY1, SCZYY2 and pneumonia caused by the new coronavirus through the database, and constructs the "compound key compound-disease target" network diagram, PPI network diagram, KEGG and GO enrichment analysis of the intersection targets of the two compound prescriptions and pneumonia caused by the new coronavirus, so as to study the core active ingredients, core action targets, core signaling pathways and core biological functions of SCZYY1 and SCZYY2 in the treatment of pneumonia caused by the new coronavirus.

[0097] The results showed that SCZYY1 had 419 therapeutic targets and 153 key active ingredients for the treatment of pneumonia caused by the new coronavirus; SCZYY2 had 452 targets and 257 key compound ingredients for the treatment of pneumonia caused by the new coronavirus. The key targets obtained by PPI were TP53, TNF, IL6, AKT1, SRC, JUN, STAT3, IFNG, IL1β, and CTNNB1. GO enrichment analysis obtained 2652 biological process entries, 284 molecular function entries, and 169 cellular component entries (SCZYY1); 2745 biological process entries, 176 molecular function entries, and 293 cellular component entries (SCZYY2). KEGG enrichment analysis obtained 256 signal pathways, and the top 40 signal pathways were screened out, mainly PI3K-AKT signaling pathway, HIF-1 signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway, IL-17 signaling pathway, etc. SCZYY2 is also related to the MAPK signaling pathway. These signal pathways are all related to the regulation of immune factors.

[0098] The PI3K-AKT signaling pathway plays an important role in cell proliferation and metabolism, and is an important pathway used by SARS-CoV-2 to complete replication. Virus-infected cells lead to overactivation of PI3K, which phosphorylates AKT, which is an important reason for the persistent infection of SARS. There is evidence that SARS-CoV-2 overactivates the PI3K-AKT signaling pathway when it infects cells through CD147 and furin, promoting viral entry through clathrin-mediated endocytosis. At the same time, studies have shown that inhibition of the PI3K / AKT signaling pathway affects NF-κB and AP-1, ultimately reducing the expression of inflammatory cytokines such as IL-6 and TNF-α. Appelberg et al. found that AKT inhibitors can significantly inhibit SARS-CV-2 infection. Therefore, AKT1 is a key target that can inhibit viral infection and the cytokine storm caused by it. SCZYY1 can also act on the MAPK signaling pathway. This signaling pathway can increase NFκB recruitment and increase the production of proinflammatory factors (Saccan et al., 2002), which is one of the main causes of cytokine storms in SARS-CoV-2 infection. Therefore, SCZYY2 may have a more significant inhibitory effect on IL6 and TNFα than SCZYY1.

[0099] Then, molecular docking was performed on the key proteins RBD (wild-type new crown and Omicron) and ACE2 for virus entry into cells, the key protein 3CLpro for virus replication, and the key proteins AKT1 and STAT3 obtained by network pharmacology analysis with the key compounds of the two compound Chinese medicines. The molecular docking results showed that the two compound prescriptions mainly inhibited viral infection by binding to the viral RBD protein, and also acted on ACE2, STAT3 and AKT1. STAT3 and AKT1 are the key upstream molecules of SARS-CoV-2 causing cytokine storm. Therefore, the two prescriptions treat pneumonia caused by the new coronavirus by inhibiting viral invasion of cells and reducing inflammatory responses. Among them, the compounds hesperidin, beta-sitosterol, naringenin and luteolin-7-o-glucoside unique to SCZYY2 are key antiviral and anti-immune compounds. The molecular docking results of the WT virus strain and the Omicron virus strain with the compound also explain the conclusion of Example 2: the effect of the two Chinese medicines on the Omicron variant is reduced, and SCZYY2 has a better effect.

[0100] like Figure 8As shown in the figure, SARS-CoV-2 infection downregulates the expression of ACE2, leading to the accumulation of AngⅡ, which binds to the AT1R receptor on the cell membrane, activates the JAK / STAT signaling pathway and induces the expression of pro-inflammatory factors. At the same time, viral RNA is recognized by TLR3 and TLR7 / 8 in the cell to activate the NF-κB signaling pathway, stimulating the expression of pro-inflammatory factors such as IL-1β, IL-18, TNFα and IL-6. At the same time, IL6 binds to the IL6 receptor (IL6R), and then binds to glycoprotein 130 to activate the JAK-STAT, AKT-PI3K and other signaling pathways, further promoting the production of pro-inflammatory factors, leading to the accumulation of inflammatory factors. These two compound Chinese medicines mainly exert anti-SARS-CoV-2 and anti-inflammatory effects by preventing the binding of S protein to ACE2 and the PI3K-AKT signaling pathway.

[0101] Example 4: Effect of compound Chinese medicine on cytokine expression

[0102] 4.1 Total RNA extraction

[0103] (1) Plant 2×10 cells in a 12-well cell culture plate. 5 / well of HEK293T cells, set up two replicate wells, and make the cell density reach about 80% overnight for infection inhibition experiment. Aspirate the culture medium and add 2mL of a mixture of three compound Chinese medicine preparations and Omicron pseudovirus with a series of concentrations. The amount of pseudovirus is MOL=1. At the same time, set up a cell control group (cells + culture medium) and a virus control group (cells + virus + culture medium). Put it in a cell culture incubator and incubate for 24 hours, then discard the supernatant and replace it with DMEM cell culture medium. Continue incubation for 36 hours and then extract RNA. After washing the cultured cell samples three times with PBS, aspirate the PBS solution, add 500μL Trizol to each well, put it on ice for 20 minutes, collect the liquid in a 1.5ml EP tube, and collect the replicate wells in the same EP tube.

[0104] (2) Add 200 μL of chloroform to a 1.5 mL enzyme-free EP tube, shake vigorously and mix for 30 seconds until it turns pink, and let it stand on ice for 15 minutes. Centrifuge at 4°C, 12,000 rpm for 15 minutes. You can see that the upper colorless aqueous liquid and the lower red liquid are clearly separated. The upper solution contains RNA. Use an enzyme-free pipette tip to carefully pipette 300 μL of the upper liquid into a new enzyme-free EP tube. Do not touch the middle white boundary and the lower liquid. Add 300 μL of isopropanol precooled at 4°C to the EP tube, mix gently for 15 seconds, and let it stand on ice for 1 hour. Centrifuge at 4°C, 12,000 rpm for 15 minutes.

[0105] (3) Take out the centrifuge tube. At this time, a trace amount of white precipitate can be seen at the bottom of the EP tube, which is RNA. Pour out the liquid in the tube and add 1 ml of pre-cooled 75% ethanol. Use an enzyme-free pipette to suspend and wash the precipitate. Be careful not to suck away the precipitate. Centrifuge at 4°C, 12000rpm for 15 minutes, and then repeat the above steps. Discard the supernatant, turn the EP tube upside down on a clean paper towel, and dry the residual liquid on the tube wall. Add an appropriate amount of DEPC water, mix gently, and dissolve the RNA.

[0106] (4) Detect RNA concentration and purity. Wash ND 2000 with 1 μL DEPC water, repeat 3 times. Load 1 μL sample into each tube and detect RNA concentration and purity. If A260 / A280 = 1.8-2.1, the purity is good. Use agarose gel electrophoresis to analyze the purity.

[0107] 4.2 Reverse transcription

[0108] (1) To remove genomic DNA, prepare a reaction mixture on ice according to the following ingredients. After gentle mixing, use a common PCR instrument and set the program to 42°C, 2 min, 4°C. The configuration of the genomic DNA removal reaction system is shown in Table 1 below:

[0109] Table 1 Genomic DNA removal reaction system configuration

[0110] Components volume 5×gDNAEraser Buffer 2.0μL gDNA Eraser 1.0μL Total RNA 1000ng <![CDATA[RNase Free dH2O]]> Up to 10μL

[0111] (2) Reverse transcription: Prepare the reaction solution on ice. After gentle mixing, use a common PCR instrument and set the program to 37°C, 15 min, 85°C, 5 s, 4°C. The configuration of the reverse transcription reaction system is shown in Table 2 below:

[0112] Table 2 qPCR system

[0113] Reagents dose The reaction solution in the previous step 10μl PrimeScript RT Enzyme MixⅠ 2.0μl RT Prime Mix 1.0μl 5×PrimeScript Buffer 2 3.0μl <![CDATA[RNase Free dH2O]]> 4.0μl

[0114] (3) The synthesized cDNA is stored at -20°C for future use or at -80°C for long-term storage.

[0115] 4.3 Real-time quantitative PCR detection of gene expression

[0116] (1) Primer design and synthesis. Primer sequences were obtained by literature search or NCBI Primer-BLAST and verified by Primer-BLAST. All primers were synthesized by Shanghai Bioengineering Co., Ltd. The primer-related information is shown in Table 3 below:

[0117] Table 3 Primer information

[0118]

[0119]

[0120] (2) The qPCR reaction system and reaction conditions are shown in Tables 4 and 5. GADPH was used as an internal reference to detect the expression of IL1b, IL6, and TNF. Primer controls (primers, water, enzyme) and negative controls (water, enzyme) were set.

[0121] Table 4 qPCR reaction system configuration

[0122]

[0123] Table 5 qPCR reaction conditions

[0124] Program Name Reaction conditions Number of cycles Pre-denaturation 95℃30s 1 Cyclic reaction 95℃10s, 60℃30s 40 Melting curve 95℃5s, 60℃60s 1

[0125] In order to determine the effects of the three compound prescriptions on the expression of immune factors induced by the Omicron pseudovirus, the expression levels of IL1b, TNFa, and IL6 at the mRNA level were detected; 24 hours after the pseudovirus infected cells, compared with the blank group, the IL1b, TNFa, and IL6 in the virus control group were significantly increased; compared with the virus control group, the IL1b, TNFa, and IL6 in the compound Chinese medicine group were significantly reduced, indicating that the three compound prescriptions inhibited the expression of proinflammatory factors IL1b, IL6, and TNFa, suggesting that the three compound Chinese medicines can regulate the immune response caused by the virus. Compared with the LH treatment group, the three factors in the SCZYY2 group were reduced to varying degrees, but there was no significant difference, which verified that SCZYY1 and SCZYY2 can regulate the large increase in cytokines caused by SARS-CoV-2 infection and have anti-immune effects.

[0126] The serial numbers of the embodiments of the present application are only for description and do not represent the advantages and disadvantages of the embodiments. The above are only preferred embodiments of the present application, and do not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made by using the contents of the present application specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. An anti-coronavirus Chinese medicine composition, characterized in that: The invention is composed of the following components in parts by weight: 10-20 parts of schizonepeta, 10-20 parts of siler, 10-20 parts of perilla leaves, 10-20 parts of dried tangerine peel, 10-20 parts of stir-fried atractylodes, 10-20 parts of tuckahoe, 10-20 parts of fructus aurantii, 10-20 parts of bupleurum, 10-20 parts of peucedanum, and 5-15 parts of roasted liquorice.

2. An anti-coronavirus Chinese medicine composition according to claim 1, characterized in that: The invention is composed of the following components in parts by weight: 15 parts of schizonepeta, 15 parts of siler, 15 parts of perilla leaves, 15 parts of dried tangerine peel, 15 parts of stir-fried atractylodes, 15 parts of tuckahoe, 15 parts of fructus aurantii, 15 parts of bupleurum, 15 parts of peucedanum, and 10 parts of roasted liquorice.

3. An anti-coronavirus Chinese medicine composition according to claim 1, characterized in that: The antiviral and anti-immune compounds in the traditional Chinese medicine composition are: hesperidin, beta-sitosterol, naringenin and luteolin-7-O-glucoside.

4. The anti-coronavirus Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition exerts an antiviral effect by inhibiting the binding of S protein to ACE2 and the PI3K-AKT signaling pathway.

5. A method for studying the mechanism of a Chinese medicine composition for resisting coronavirus, characterized in that: The method for detecting the anti-coronavirus Chinese medicine composition according to any one of claims 1 to 4 comprises the following steps: S1. Construction of pseudovirus: The original strain and its variant pseudovirus carrying green fluorescent protein GFP were packaged using the HIV-1 three-plasmid system, concentrated using the PEG8000 method, and the infection ability and titer of the pseudovirus were verified by infection experiments; S2. Evaluation of the anti-pseudovirus infection effect of the Chinese medicine combination: The cytotoxicity of the compound Chinese medicine was detected by the CCK8 method to determine the safe action time and concentration; through the infection inhibition experiment, two pseudoviruses and three compound drugs were co-cultured respectively, and the number of green fluorescent cells was observed under a fluorescence microscope to determine the effect of the compound Chinese medicine on the virus infection activity; S3. Exploration of the potential mechanism of Chinese herbal medicine combination against coronavirus: exploring the potential mechanism through network pharmacology and molecular docking technology; S4. Real-time fluorescence quantitative PCR technology was used to detect the anti-inflammatory effect of drugs on pseudovirus infection: total cell RNA was extracted using the trizol RNA extraction method, and after reverse transcription, fluorescence quantitative PCR technology was used to detect the effects of the three compounds on the expression of IL1b, IL6, and TNF.

6. The method for studying the mechanism of anti-coronavirus effect of traditional Chinese medicine according to claim 5, characterized in that: In step S1, the three-plasmid system includes pCDH-GFP, psPAX2 and the envelope plasmid of spike protein.

7. The method for studying the mechanism of anti-coronavirus effect of traditional Chinese medicine according to claim 5, characterized in that: In step S1, the concentrated virus titer is calculated as follows: The titer of the concentrated virus = the number of fluorescent cells in the field of view × dilution factor × 1000 mL / the volume of the original concentrated virus added.

8. The method for studying the mechanism of anti-coronavirus effect of traditional Chinese medicine according to claim 5, characterized in that: The step S3 specifically includes the following sub-steps: S31. Use TCMSP to determine the target of the compound, and GeneCards, TTD, and OMIM databases to determine the targets related to pneumonia caused by the new coronavirus; S32. Use Cytoscape to draw a "key compound-target network diagram" to obtain the core drug small molecule compounds; S33, use PPI to generate protein interaction network diagram and obtain core targets; S34, GO and KEGG enrichment analysis was performed using the DAVID database; S35. Use AutoDock to perform molecular docking of key small molecule compounds and screened key targets.

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