CN115778941A

CN115778941A - Anti-neocoronavirus combined pharmaceutical composition and application thereof

Info

Publication number: CN115778941A
Application number: CN202211448559.1A
Authority: CN
Inventors: 刘慧迪; 刘树林; 吴昊; 张孟春; 林慧慧; 陈航
Original assignee: Harbin Medical University
Current assignee: Harbin Medical University
Priority date: 2022-11-18
Filing date: 2022-11-18
Publication date: 2023-03-14

Abstract

The invention relates to a combined drug composition for resisting new coronavirus and application thereof, belonging to the technical field of biological medicine. In order to solve the problems of poor effectiveness and large toxic and side effects of the existing anti-new coronavirus vaccines and medicines, the invention provides an anti-new coronavirus combined medicine composition, which comprises an effective amount of CBD and an effective amount of EGCG. The anti-new coronavirus medicine is used for inhibiting new coronavirus invasion and re-invasion of human cells. Experiments prove that after the CBD and the EGCG are jointly used, the compound has good synergistic effect of resisting the invasion of the new coronavirus. The inhibitor can play a role in inhibiting the cell invasion process of the new coronavirus S-pseudovirus and the expression of spike protein of the new coronavirus after invasion under the safe concentration of the two medicines, realizes the multi-level inhibition effect on the invasion and later re-invasion of the virus, and simultaneously lightens the side effect of reducing ACE2 protein by CBD, thereby having good practical application value.

Description

Anti-neocoronavirus combined pharmaceutical composition and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a new coronavirus resistant combined medicine composition and application thereof.

Background

The excessive reaction of the immune system of the organism caused by the infection of Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) leads to Severe inflammation of the organism, and leads to the occurrence of mucosal inflammation, lung injury and multi-organ failure of patients with novel Coronavirus infection (Coronavir Disease 2019, CODVID-19).

Spike protein (S) on the surface of SARS-CoV-2 plays an important role in the process of first invasion of virus and re-invasion of virus infected cells to adjacent cells, wherein the S1 subunit contains a Receptor Binding Domain (RBD) which is responsible for recognizing and binding angiotensin converting enzyme 2 (ACE2) protein receptor on the surface of human cells, and then the virus is anchored on the surface of the cells through the S2 subunit to generate membrane fusion, and mediate the virus to enter the cells and carry out subsequent processes such as integration, replication, maturation release and the like. A large number of researches show that ACE2 plays a key role in the process of SARS-CoV-2 infection of human body, the expression level of ACE2 is an important factor influencing the susceptibility of SARS-CoV-2, and the infection of virus can also cause the change of host ACE2 level. The roles of ACE2 in different stages of the disease course of novel coronavirus infection are different, and the maintenance of normal expression of ACE2 has important effects on regulating body immunity and avoiding pneumonia aggravation although the ACE2 mediates virus invasion. ACE2 is one of important regulatory proteins of human body, widely distributed on cell surface of organs such as heart, kidney, lung, testis and liver, and is involved in regulating cell proliferation, blood pressure change, fluid balance and inflammatory reaction. Abnormalities in the expression level of ACE2 can lead to serious complications after viral infection, aggravate the body's cytokine storm, and destroy the renin-angiotensin-aldosterone system (RAAS), thereby further inducing acute respiratory distress syndrome.

Current treatments for COVID-19 include novel coronavirus vaccines and traditional antiviral drugs for prevention of SARS-CoV-2 infection. Because the mutation speed of SARS-CoV-2 is very fast, the effective period of the existing vaccine is shorter, and the effect of preventing infection can not achieve better effect.

Conventional antiviral drugs such as Chloroquine (CQ) and its derivatives hydroxychloroquine associated with viral invasion, reidesivir (remdesivir), lopinavir, ritonavir anchoring virus nonstructural proteins, and receptor blockers associated with body immunity such as Interleukin-6 (The Interleukin, IL-6) receptor blocker tolizumab, salizumab, and The like. At the same time, the glucocorticoid drug dexamethasone has also been shown to play an important role in combating COVID-19.

The drugs put into use so far are mostly applied to symptomatic treatment, but the results in clinical trials show that the effect of the drugs on COVID-19 is limited or even ineffective. Meanwhile, in the later stage of development of COVID-19, the using effect of a plurality of medicines is often poor, and a medicine-resistant mutant strain can appear or has larger toxic and side effects, thereby causing new threat to human body. For example, although the traditional antiviral drug chloroquine is administered for treatment at the first time, the therapeutic effect is still questionable due to the defects of narrow therapeutic window, strong toxic and side effects, and the like which may cause cardiac complications. While the other clinically used medicament, namely the Redeciclovir, can cause gastrointestinal reactions such as nausea, vomiting and the like, and causes physical burden to patients. Meanwhile, the complexity of the reaction caused by SARS-CoV-2 acting on human body causes the bottleneck of the current research of specific drugs aiming at COVID-19, and the solution is urgently needed to find safer and more effective preventive and therapeutic drugs.

Disclosure of Invention

The invention provides a new coronavirus resistant combined pharmaceutical composition and application thereof, aiming at solving the problems of poor effectiveness and large toxic and side effects of the existing new coronavirus resistant vaccine and medicine. The existing research on anti-SARS-CoV-2 medicine mostly focuses on inhibiting virus RNA replication and enhancing organism immunity, and the research on medicine in the virus invasion process is less. The invention provides a direction for combining natural medicines and resisting COVID-19 from the angle of inhibiting the process of SARS-CoV-2 invading cells.

The technical scheme of the invention is as follows:

a pharmaceutical composition for treating new crown virus comprises effective amount of Cannabidiol (CBD) and epigallocatechin-3-gallate (EGCG).

In the technical scheme of the invention, CBD is shown as a structural formula (1), EGCG is shown as a structural formula (2):

furthermore, the safe and effective concentration range of CBD in the drug combination composition is 5-15 mu mol/L, and the safe and effective concentration range of EGCG is 10-50 mu mol/L.

Further, the molar ratio of the effective amount of CBD to the effective amount of EGCG in the pharmaceutical combination composition is 1.

Furthermore, the optimal concentration of CBD in the combined medicine composition is 10 mu mol/L, and the optimal concentration of EGCG is 30 mu mol/L.

Furthermore, the combined medicine composition also comprises a pharmaceutically acceptable carrier or excipient.

Further, the pharmaceutically acceptable carrier comprises a liposome, a micelle, a dendrimer, a microsphere or a microcapsule.

Furthermore, the dosage form of the combined drug composition is tablets, granules, capsules, solutions, pills, liposome preparations or nanoparticle preparations.

Furthermore, the combined medicine composition is a single compound preparation or is divided into two separate preparations of an EGCG preparation and a CBD preparation; the two separate formulations are for simultaneous, separate or sequential administration.

An application of a pharmaceutical composition for resisting new coronavirus in the preparation of anti-new coronavirus medicines is provided.

Furthermore, the anti-new coronavirus medicine is used for inhibiting new coronavirus invasion and re-invasion of human cells.

The invention has the beneficial effects that:

experiments prove that after the CBD and the EGCG are jointly used, the compound has good synergistic effect of resisting the invasion of the new coronavirus. The inhibitor can play a role in inhibiting the cell invasion process of the new coronavirus S-pseudovirus and the expression of spike protein of the new coronavirus after invasion under the safe concentration of the two drugs, and realizes the multi-level inhibition effect on the invasion and later re-invasion of the virus, thereby having good practical application value.

The invention combines CBD and EGCG for administration, overcomes the defects of high effective concentration of CBD and certain toxic and side effect, eliminates the possibility that the combined use of CBD and EGCG can generate interaction to reduce the respective SARS-CoV-2 infection resisting effect of CBD and EGCG, can perform function complementation on different levels after the combination of the two medicines, inhibits cells infected by new corona s-pseudovirus, can inhibit the first invasion of the virus and the re-invasion of the virus by reducing spike protein expression, and shows good multi-level synergistic anti-new corona virus treatment effect. Meanwhile, the combined medication reduces the side effect of CBD inhibiting human ACE2 protein expression, restores the normal ACE2 protein expression level of the organism, and avoids the possibility of aggravation of complications and symptoms caused by the change of the ACE2 expression level, such as acute respiratory distress syndrome, severe lung failure, thromboembolism, glycolipid metabolic disturbance, vascular injury, reproductive and nervous system influence and the like. The combined application of CBD and EGCG provides a research direction for natural products to resist novel coronavirus infection.

Drawings

FIG. 1 is a graph showing the results of ACE2 protein of 293T-OE cells in Western Blot method in example 1;

FIG. 2 is a bar graph of the cell activity of 293T-OE cells in the CCK8 method of example 1 under optimal concentration conditions for administration of CQ, CBD and EGCG;

FIG. 3 is a green fluorescence micrograph showing that the S-pseudoviral vector carrying a GFP fluorescent tag of example 2 infects 293T-OE cells under the condition of optimal concentration of administered drugs CQ, CBD, EGCG;

FIG. 4 is a bar graph of the cell invasion rate of 293T-OE cells infected by S-pseudoviral vector carrying GFP fluorescent tag in example 2 under optimal concentration conditions for administration of drugs CQ, CBD, EGCG;

FIG. 5 is a graph showing the results of the S protein of 293T-OE-S cells in the Western Blot method in example 3;

FIG. 6 is the result chart of the S protein and ACE2 protein of 293T-OE-S cells under the action of optimal concentration drugs CQ, CBD and EGCG in Western Blot method in example 3;

FIG. 7 is the result chart of the S protein of 293T-OE-S cells under the action of optimal concentration of drugs CQ, CBD and EGCG in the qRT-PCR method of example 4;

FIG. 8 is the result chart of ACE2 protein of 293T-OE-S cells under the action of optimal concentration drugs CQ, CBD and EGCG in the qRT-PCR method of example 4;

FIG. 9 is a schematic diagram of the binding of the inhibitory virus S to ACE2 protein with CBD occupying the ACE2 protein binding site in molecular docking of example 5;

FIG. 10 is a schematic diagram of the binding of virus S and ACE proteins inhibited by CBD occupying the binding site of virus S protein in molecular docking of example 5;

FIG. 11 is the schematic diagram of the binding of EGCG to ACE2 protein binding site and the inhibition of virus S to ACE protein in molecular docking in example 5;

FIG. 12 is the schematic diagram of the binding of EGCG to the binding site of the virus S protein and the inhibition of the virus S protein to ACE protein in molecular docking in example 5;

FIG. 13 is a schematic representation of the interaction of ACE2 protein molecules with CBDs in molecular docking according to example 5;

FIG. 14 is a schematic diagram showing the interaction between ACE2 protein molecules and EGCG in molecular docking in example 5;

FIG. 15 is a schematic diagram of the interaction of SARS-CoV-2S protein molecule and CBD in molecular docking of example 5;

FIG. 16 is a schematic diagram of the interaction of SARS-CoV-2S protein molecule and EGCG in molecular docking of example 5;

FIG. 17 is a schematic diagram of the interaction of SARS-CoV-2S protein molecule with CBD and EGCG in molecular docking of example 5;

FIG. 18 is a bar graph of the optimal concentration of drug versus the activity of cells for the combination of CBD and EGCG in the CCK8 method of example 6;

FIG. 19 is a green fluorescence microscopy result of 293T-OE cells infected by GFP fluorescent tagged S-pseudoviral vector of example 7 under the condition of optimal concentration of administered drugs of Control, CQ, CBD, EGCG and combination of CBD and EGCG;

FIG. 20 is a bar graph of the cell invasion rates of 293T-OE cells infected by the S-pseudoviral vector carrying GFP fluorescent tags in example 2 under the conditions of optimal concentrations of the administered drugs, control, CQ, CBD, EGCG and the combination of CBD and EGCG;

FIG. 21 is a graph showing the results of the protein S and ACE2 protein of 293T-OE-S cells in Western Blot at optimal concentrations in Control, CBD, EGCG and the combination of CBD and EGCG.

Detailed Description

The technical solutions of the present invention are further described below with reference to the following examples, but the present invention is not limited thereto, and any modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention. The process equipment or apparatus not specifically mentioned in the following examples are conventional in the art, and if not specifically mentioned, the raw materials and the like used in the examples of the present invention are commercially available; unless otherwise specified, all technical means used in the examples of the present invention are conventional means well known to those skilled in the art.

The invention simulates a plurality of processes of infecting human cells by the virus through the constructed new coronavirus S-pseudovirus and a host cell model expressing S protein after virus infection so as to test the combined action of CBD and EGCG in the process of resisting virus infection. Firstly, the influence of natural drugs on the first invasion of the novel coronavirus into the normal cells of a human body is detected by simulating the process of infecting the novel coronavirus into the cells of the human body by using SARS-CoV-2 spike-protein pseudovirus (S-pseudovirus) and human kidney epithelial ACE2 overexpression cells (293T-OE). On the other hand, a post-infection host cell model (293T-OE-S) which is constructed by lentivirus and can express a novel coronavirus S protein is utilized to detect the effect of each medicament on the expression of the virus S protein and the human ACE2 protein, and the influence of CBD and EGCG on re-invasion of virus from infected cells to adjacent cells mediated by the S protein is evaluated. The feasibility of combining the two medicines is verified through molecular docking, and then the combined medicines are used to test the effect of combining the CBD and the EGCG.

Example 1

In this example, the optimal concentration and safe and effective range of the CBD and EGCG administration are determined by using ACE2 over-expressed human kidney epithelial cell line 293T (293T-OE) cells through a cell proliferation-toxicity detection kit (cell counting kit-8, CCK8) method.

This example 293T-OE cells were treated with CQ (positive drug chloroquine) at concentrations of 2, 4, 6, 8, 10, 12 and 14. Mu. Mol/L, 293T-OE cells were treated with CBD at concentrations of 2.5, 5, 7.5, 10, 12.5, 15 and 17.5. Mu. Mol/L, 293T-OE cells were treated with EGCG at concentrations of 10, 20, 30, 40, 50, 60, 70 and 80. Mu. Mol/L, and drug optimal concentrations were screened; the cytotoxic concentrations (CC 50) were obtained by treating 293T-OE cells with CQ at concentrations of 4, 8, 12, 16, 20, 24, 28 and 32. Mu. Mol/L, by treating 293T-OE cells with CBD at concentrations of 10, 20, 30, 40, 50 and 60. Mu. Mol/L, and by treating 293T-OE cells with EGCG at concentrations of 20, 40, 60, 80, 100, 120, 140 and 160. Mu. Mol/L.

The implementation method comprises the following steps: cells were passaged normally and cell density was adjusted to 10 ⁴ And (4) inoculating the cells into a 96-well plate at a concentration of 100 mu L per well, culturing the cells at 37 ℃ for 24h, treating the cells with CQ, CBD and EGCG at the concentrations, and adding DMSO at the same concentration into the cells in the control group as a control. After adding drugs for 0h, 24h, 48h and 72h, preparing 10% CCK8 solution in dark, culturing at 37 ℃, observing the state of each group of cells, and measuring the absorbance value at 450nm by using an enzyme-labeling instrument. After data processing, graphpadprism 9 is used for drawing to obtain the optimal concentration, the safe and effective range and the drug toxicity of the drug.

According to the 293T-OE cell state and the CCK8 detection result under different concentrations, through repeated experiments, the effective concentration range of CBD is 5-15 mu mol/L and the optimal concentration is 10 mu mol/L, the effective concentration range of EGCG is 10-50 mu mol/L and the optimal concentration is 30 mu mol/L.

As a result: FIG. 1 and FIG. 2 are the Western blot result chart, the optimal concentration of the drug-cell activity histogram of the constructed 293T-OE cells. As can be seen from the figure, the safe and effective concentration range of CQ single administration is 2-8 mu mol/L, and the optimal concentration is 5 mu mol/L; the safe and effective concentration range of the single CBD administration is 5-15 mu mol/L, and the optimal concentration is 10 mu mol/L; the safe and effective concentration range of single EGCG administration is 10-50 mu mol/L, and the optimal concentration is 30 mu mol/L.

Example 2

In this example, an S-pseudovirus vector carrying a Green Fluorescent Protein (GFP) fluorescent tag was constructed, and the inhibitory effect of EGCG and CBD on invasion of a new coronavirus was examined.

The S-pseudovirus vector which is constructed by the lentivirus vector and carries the GFP fluorescent label has the characteristics of high safety, good stability, strong operability and the like, and is an effective common tool for researching the process of SARS-CoV-2 invading normal cells at a low biological safety level.

The implementation method comprises the following steps: in order to observe the effect of EGCG and CBD on the invasion process of the novel coronavirus S-pseudovirus at the optimal concentration, in the present example, EGCG and CBD were applied to 293T-OE cells respectively, CQ was used as a positive control drug, and after 24h of drug treatment, the cells were infected with S-pseudovirus with GFP fluorescent tag for 24h. After the culture medium was replaced, the culture was continued for 48 hours, and the expression of GFP was observed under an inverted fluorescence microscope to evaluate the invasion efficiency of S-pseudovirus. The CQ concentration used in this example was 4. Mu. Mol/L, the CBD concentration was 10. Mu. Mol/L and the EGCG concentration was 30. Mu. Mol/L.

As a result: FIGS. 3 and 4 are the photographs (100 μm) of the result of the invasion of cells by the S-pseudoviral vector carrying the GFP fluorescent tag under different administration conditions (Control, CQ, CBD, EGCG) and the histogram of the cell invasion rate. FIG. 3 shows brightly the status of all adherent cells after pseudovirus invasion with different drugs, and the fluorescence field shows cells that invade with pseudovirus carrying GFP and successfully fluoresce green under excitation light. The result shows that the inhibition effect of CBD on virus invasion is similar to that of CQ serving as a positive control drug, and EGCG has certain inhibition effect but has insignificant effect.

CBD has a more obvious effect of inhibiting invasion of new crown S-pseudovirus, and EGCG has a certain inhibition effect but is less obvious than CBD.

Example 3

This example uses a human renal epithelial ACE2 overexpressing cell line 293T cells (293T-OE-S) capable of expressing a novel coronavirus S protein to mimic the expression of the virus S protein in infected host cells, and the effect of EGCG and CBD on inhibiting the expression levels of the S protein and ACE2 protein was examined by Western Blot.

The implementation method comprises the following steps: the 293T-OE-S cell total protein after the medicaments act for 72 hours is collected by a conventional method, and the steps of glue preparation, electrophoresis, membrane transfer, primary antibody and secondary antibody incubation, membrane washing, color development and imaging analysis are carried out after the concentration of a protein sample is determined.

As a result: FIGS. 5 and 6 are a Western Blot result chart of the constructed 293T-OE-S cell and Western Blot result charts of S protein and ACE2 protein under the action of optimal concentration drugs CQ, CBD and EGCG. Through repeated experiments, the expression of the S protein of the cell model after the EGCG treatment group is infected is obviously reduced, and the expression of the ACE2 protein of the cell surface receptor is slightly increased compared with that of a control group. Compared with EGCG-treated group, the CBD-treated group has no obvious reduction of S protein expression, but has obviously reduced ACE2 protein expression.

At the protein expression level, EGCG can down-regulate the expression of S protein but slightly up-regulate the expression of ACE2 protein, while CBD has no obvious effect on S protein but can significantly down-regulate the expression of ACE2 protein.

Example 4

This example uses 293T-OE-S cells to analyze the effects of EGCG and CBD on spike and ACE2 gene mRNA expression by real-time quantitative PCR (qRT-PCR).

The implementation method comprises the following steps: extracting total RNA from a cell sample, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification by using a specific primer, analyzing data by adopting a 2-delta CT method, and detecting the transcription level of the mRNA.

As a result: FIGS. 7 and 8 are the results of qRT-PCR of spike and ACE2 genes of 293T-OE-S after 48h of optimum concentrations of CQ, CBD and EGCG. Similar to the Western-Blot result, the qRT-PCR result shows that the mRNA transcription of the spike gene of the EGCG treatment group is obviously reduced compared with that of the control group, the mRNA transcription of the ACE2 gene is obviously improved, and the mRNA transcription change of the spike gene of the CBD treatment group is not obvious but the mRNA transcription of the ACE2 gene is obviously reduced.

At the transcriptional level, the transcriptional impact of CBD on mRNA of spike gene was insignificant and EGCG down-regulated mRNA transcription of spike gene.

Example 5

This example simulates the binding of CBD and EGCG to ACE2 and S proteins by molecular docking

The experimental method comprises the following steps: the 3D structure of CBD and EGCG was derived from the NCBI PubChem database. The structure optimization and the prediction of the protonation state of the S protein and the ACE2 protein are carried out by using MOE (2022 edition) software, and the docking site of the S protein RBD is determined by literature research. The determination of the ACE2 docking site is performed by scanning the binding surface of ACE2 and S protein through software. And (3) carrying out molecular docking of the target protein and the drug molecules by using MOE software.

As a result: FIGS. 9-12 are, in order, schematic diagrams of CBD occupying the ACE2 protein binding site, CBD occupying the viral S protein binding site, EGCG occupying the ACE2 protein binding site, and EGCG occupying the viral S protein binding site to inhibit binding of the viral S protein to the ACE protein, and FIGS. 13-17 are, in order, schematic diagrams of the binding patterns of ACE2 protein coacting with CBD and EGCG, respectively, S protein with CBD and EGCG, respectively, and S protein with CBD and EGCG. The score of the optimal interaction model of the SARS-CoV-2S protein molecule and the CBD is-5.22 kcal/mol, the score of the optimal interaction model of the SARS-CoV-2S protein molecule and the EGCG is-5.88 kcal/mol, the score of the optimal interaction model of the human ACE2 receptor and the CBD is-6.78 kcal/mol, and the score of the optimal interaction model of the human ACE2 receptor and the EGCG is-6.87 kcal/mol. The smaller the score, the stronger the interaction capacity.

The SARS-CoV-2S protein has two docking conformational distribution sites in the first 10 best binding modes of the two drug molecules. Since the binding affinity of EGCG to S protein is stronger than that of CBD, when two drugs are combined, after the optimal binding site is inclined to be combined by EGCG, CBD may be combined to another binding site to exert the synergistic effect on the drug effect. The molecular docking results indicate the feasibility of using the CBD and the EGCG together.

Example 6

In this example, the concentration of CBD and EGCG in combination was verified by the CCK8 method.

The experimental method comprises the following steps: 293T-OE cells were passaged normally, adjusting cell density to 10 ⁴ The cells were inoculated into a 96-well plate at a concentration of 100. Mu.L per well, and cultured at 37 ℃ for 24 hours. The CBD and EGCG are combined to act on the cells at the optimal concentration, wherein the concentrations of the CBD and the EGCG in the combined drug are respectively 10 mu mol/L and 30 mu mol/L. After the cells are respectively treated by the medicine for 24h, 48h and 72h, the cell activity is detected by a CCK8 method.

As a result: FIG. 18 is a bar graph of the optimal concentration of drug for the combination of CBD and EGCG-cell activity, and the activity of the cells after 24h, 48h and 72h of culture under the drug concentration of the combination is detected, and the drug concentration has no obvious influence on the activity of the cells.

The safe concentrations for the administration of CBD in combination with EGCG were 10. Mu. Mol/L CBD and 30. Mu. Mol/L EGCG.

Example 7

In the embodiment, the effect of the combined drug of EGCG and CBD in inhibiting the invasion process of the new corona S-pseudovirus is detected through an S-pseudovirus invasion test.

The implementation method comprises the following steps: s-pseudovirus invasion assay As above, the concentrations of CBD and EGCG in combination used to treat 293T-OE in this example: CBD is 10 mu mol/L, EGCG is 30 mu mol/L.

As a result: FIGS. 19 and 20 are a green fluorescence (100X) and a histogram of cell invasion rates of 293T-OE cells treated with S-pseudovirus invasion drugs (CQ, CBD, EGCG and combination of CBD and EGCG). The results show that the inhibition ability of the combination of CBD and EGCG on virus invasion is similar to that of a positive control drug CQ.

The combined medication of the CBD and the EGCG can inhibit the invasion of new crown S-pseudoviruses and has no obvious influence on the function of the CBD in inhibiting the invasion of the S-pseudoviruses.

Example 8

In this example, the effect of EGCG in combination with CBD on the level of inhibiting the expression of S protein and ACE2 protein was examined by Western Blot method.

The implementation method comprises the following steps: western Blot procedure is as above. The concentrations of CBD and EGCG in combination used in this example were: CBD is 10 mu mol/L, EGCG is 30 mu mol/L.

As a result: FIG. 21 is a graph showing Western Blot results of protein S and protein ACE2 of 293T-OE-S under the effect of optimum concentrations of drug CBD, EGCG and combination of CBD and EGCG. The experimental result shows that after the CBD and the EGCG are combined, the protein expression level of spike is obviously reduced, and the protein expression of ACE2 is similar to that of a control group.

On the protein expression level, the combined administration does not affect the inhibiting effect of EGCG on S protein expression, and the ACE2 expression is not affected.

The invention firstly utilizes 293T-OE cells and chloroquine as a positive control drug to detect the cytotoxicity of CBD and EGCG by a CCK8 method, and determines that the safe action concentration ranges of the CBD and the EGCG are respectively 5-15 mu mo/L and 10-50 mu mol/L, and the optimal drug concentrations are respectively 10 mu mo/L and 30 mu mol/L. The 293T-OE cells are infected with an S-pseudovirus vector carrying a GFP fluorescent tag, and the invasion of the virus is evaluated by detecting the expression level of GFP. The result shows that CBD can obviously inhibit the invasion of S-pseudovirus, and the action effect is similar to that of chloroquine. Meanwhile, 293T-OE-S cells which are constructed by lentiviruses and can express virus spike proteins are utilized to simulate the expression of cell virus S proteins after host infection, the medicines (CQ, CBD and EGCG) are acted on the cells, and the influence of each medicine on the transcription and expression levels of the novel coronavirus S proteins and the ACE2 proteins is researched. qRT-PCR results show that EGCG can remarkably reduce the mRNA level of spike of the novel coronavirus, while CBD has no obvious influence on the mRNA level of spike but can remarkably reduce the mRNA level of ACE 2; western Blot detection results show that EGCG obviously down-regulates the expression level of S protein, and CBD obviously down-regulates the expression level of ACE2 protein of cells.

In view of the functional complementarity of the two drugs, the interaction of CBD and EGCG with human ACE2 and SARS-CoV-2S protein molecules is simulated through molecular docking, and the results show that EGCG and CBD share two advantageous binding modes with S protein, and the binding affinity of EGCG is stronger than that of CBD. When EGCG is bound to the optimal binding site, CBD tends to bind to another site to exert a synergistic effect of inhibiting the binding of S protein to ACE2 receptor when the two drugs are used in combination. Based on the results, the combination of the CBD and the EGCG can inhibit virus-infected cells at multiple levels, namely the combination does not influence the inhibition effect of the CBD on S-pseudovirus first-time invading cells, and simultaneously the EGCG can play the inhibition effect on virus S protein expression in infected cells to inhibit invaded viruses from invading adjacent cells again.

Based on the importance of the ACE2 protein level in the body and its complexity in the pathogenic process of viruses, changes in the ACE2 protein expression level can cause complex changes in the body. Abnormal ACE2 expression can cause severe cytokine storm in the body to cause acute respiratory distress syndrome, other diseases of the respiratory system such as severe lung failure, and complications of the cardiovascular system such as thromboembolism, and influence the normal functions of the reproductive system and the nervous system, so that the stability of the ACE2 level in the body has important significance. Compared with a control group, the expression level of ACE2 in the combination group of the CBD and the EGCG is not obviously changed, which indicates that the combination of the CBD and the EGCG can avoid the expression inhibition of the ACE2 caused by the single medication of the CBD and reduce the possibility of side effects and complications caused by abnormal expression level of the ACE 2.

The invention proves that the combined administration of EGCG and CBD can inhibit the first invasion of S-pseudovirus and inhibit the process of virus re-invasion of adjacent cells by inhibiting the expression of virus S protein in infected cells compared with the single administration of the EGCG and CBD. Compared with the single drug of the EGCG and the CBD, the combined drug of the EGCG and the CBD more effectively inhibits the virus invasion and subsequent re-invasion processes at multiple levels, reduces the efficiency of the novel coronavirus in the invasion and re-invasion processes, can reduce the side effect of inhibiting the ACE2 expression level of normal cells of an organism by the CBD, and finally realizes the purpose of safely and effectively resisting the new coronavirus infection.

Claims

1. A pharmaceutical composition for treating new coronavirus which comprises an effective amount of cannabidiol and an effective amount of epigallocatechin-3-gallate.

2. The anti-neocoronavirus combination pharmaceutical composition according to claim 1, wherein the safe and effective concentration range of CBD in the combination pharmaceutical composition is 5-15 μmol/L, and the safe and effective concentration range of EGCG is 10-50 μmol/L.

3. The anti-neocoronaviruse combination pharmaceutical composition of claim 1, wherein the molar ratio of the effective amount of CBD to the effective amount of EGCG in the combination pharmaceutical composition is 1.

4. The anti-neocoronaviruse combination pharmaceutical composition of claim 1, wherein the optimal concentration of CBD is 10 μmol/L and the optimal concentration of EGCG is 30 μmol/L.

5. The anti-neocoronaviral combination according to any one of claims 1 to 4, wherein said combination further comprises a pharmaceutically acceptable carrier or excipient.

6. The anti-neocoronavirus combination pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable carrier comprises a liposome, a micelle, a dendrimer, a microsphere, or a microcapsule.

7. The anti-neocoronaviruse pharmaceutical combination composition according to claim 6, wherein the pharmaceutical combination composition is in the form of tablet, granule, capsule, solution, pill, liposome or nanoparticle.

8. The anti-neocoronaviruse pharmaceutical combination according to claim 7, wherein the pharmaceutical combination is a single compound preparation or two separate preparations, namely an EGCG preparation and a CBD preparation; the two separate formulations are for simultaneous, separate or sequential administration.

9. Use of a pharmaceutical combination according to any one of claims 1 to 8 for the treatment of a new coronavirus infection.

10. The use of the anti-neocoronavirus combination pharmaceutical composition of claim 9 for the preparation of an anti-neocoronavirus medicament, wherein the anti-neocoronavirus medicament is used for inhibiting invasion and re-invasion of human cells by a neocoronavirus.