阅读说明：本技术 3-表齐墩果酸在制备预防和/或治疗新冠病毒感染的药物中的用途 (Application of 3-epioleanolic acid in preparation of medicine for preventing and/or treating new coronavirus infection ) 是由 张晗 苗琳 张敏 王彧 陈璐 张俊华 王跃飞 李霖 闫慧敏 杨柳 于 2021-10-08 设计创作，主要内容包括：本申请提供了3-表齐墩果酸在制备预防和/或治疗新冠病毒感染的药物中的用途。3-表齐墩果酸能够抑制新冠病毒S蛋白和ACE2结合,从而能够用于预防和/或治疗新冠病毒感染,进而用于制备预防和/或治疗新冠病毒感染的药物。进一步地,包含3-表齐墩果酸的药物组合物,同样能够用于制备预防和/或治疗新冠病毒感染的药物。(The application provides application of 3-epioleanolic acid in preparing a medicament for preventing and/or treating new coronavirus infection. The 3-epioleanolic acid can inhibit the combination of the S protein of the new coronavirus and ACE2, so that the 3-epioleanolic acid can be used for preventing and/or treating the infection of the new coronavirus, and further can be used for preparing a medicine for preventing and/or treating the infection of the new coronavirus. Further, the pharmaceutical composition comprising 3-epioleanolic acid can also be used for preparing a medicament for preventing and/or treating new coronavirus infection.)

1.3-use of oleanolic acid in preparation of medicine for preventing and/or treating new coronavirus infection.

2. The use of claim 1, wherein the neocoronavirus infection comprises at least one of pneumonia and acute respiratory distress syndrome caused by a neocoronavirus infection.

3. The use according to claim 1, wherein 3-epioleanolic acid prevents and/or treats neocoronavirus infection by inhibiting the binding of neocoronavirus S protein to host ACE 2.

4. A pharmaceutical composition for preventing and/or treating a neocoronavirus infection, comprising 3-epioleanolic acid.

5. The pharmaceutical composition according to claim 4, wherein the 3-epioleanolic acid is provided in the form of a monomer, or in the form of a plant extract containing the same.

6. The pharmaceutical composition of claim 5, wherein the plant extract is selected from at least one of a Han peach leaf extract, a Scutellaria barbata extract, and a verbena extract.

7. The pharmaceutical composition of any one of claims 4-6, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable carrier or excipient is selected from at least one of a solvent, a diluent, a disintegrant, a precipitation inhibitor, a surfactant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonicity agent, a thickener, an emulsifier, a preservative, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a colorant, a flavoring agent, a sweetener, an ion exchanger, a mold release agent, a coating agent, a flavoring agent, or an antioxidant.

9. The pharmaceutical composition of any one of claims 4-6, wherein the pharmaceutical composition is formulated as any one of a powder, a tablet, a capsule, a pill, a drop pill, an emulsion, a suspension, or a tincture.

Technical Field

The application relates to the technical field of medicines, in particular to application of 3-epioleanolic acid in preparing a medicine for preventing and/or treating new coronavirus infection.

Background

The new type coronavirus (SARS-CoV-2, new coronavirus for short) has strong infectivity and high pathogenicity, and poses a great threat to human health. SARS-CoV-2 is a novel beta coronavirus, and its transmission mode is presumed to be that it is transmitted by animal and then interpersonal transmission; respiratory droplets and intimate contact transmission are the primary transmission pathways, and the potential for transmission via aerosols exists in relatively closed environments with prolonged exposure to high concentrations of aerosols. With the rapid development of epidemic situations, the development of drugs against novel coronaviruses is imminent.

SARS-CoV-2 virus can infect cells by combining spike glycoprotein (spike, S protein) with respiratory angiotensin converting enzyme (ACE2), the S protein on the surface of the novel coronavirus is a key protein of corresponding receptor on virus recognition target cells, and the S protein is effectively combined with ACE2 on the surface of human cells, which causes novel coronavirus pneumonia (COVID-19, new coronary pneumonia for short) and Acute Respiratory Distress Syndrome (ARDS), and part of patients have acute changes in illness in a short period and have multi-organ failure or even death. The S protein of the novel coronavirus is divided into two subunits of S1 and S2, the S1 subunit comprises an N-terminal domain (NTD) and a C-terminal domain (CTD), wherein the CTD has the function of receptor recognition and binding and is also called a Receptor Binding Domain (RBD); ACE2 is a peptidase belonging to type I transmembrane protein, and is mainly distributed in type II alveolar cells in lung tissues, and is distributed in small amount in type I alveolar cells, airway epithelial cells, fibroblasts, endothelial cells and macrophages; the RBD domain on the S1 subunit is responsible for binding to ACE2 on the host cell, further fusing with the cell membrane of the host to infect humans. Therefore, blocking the binding of the S protein and ACE2 is one of the important directions for treating COVID-19, and the aim of preventing virus infection of human body is achieved by blocking or competitively inhibiting the binding of the S protein and ACE 2.

The epidemic situation of pneumonia caused by SARS-CoV-2 is better controlled than the high outbreak of SARS-CoV virus epidemic situation in 2003, but the lack of specific therapeutic drugs is still a big problem. Although lopinavir and ritonavir have been used in the treatment of clinical neocoronary pneumonia, the search for new safe specific drugs is urgent due to their great side effects. The method and the device for screening and identifying the anti-SARS-COV-2 traditional Chinese medicine by blocking S protein RBD or ACE2 and/or inhibiting the combination of the two as targets.

At present, the research reports on 3-epi-oleanolic acid are rare, and the existing research shows that the 3-epi-oleanolic acid is possibly a receptor agonist of glucagon-like peptide-1 (CLP-1) and has the potential effect of inhibiting obesity; can stimulate the contraction of the uterine muscle strips of the guinea pig, and has good uterine contraction activity; also has anti-inflammatory effect; however, other pharmacological actions of 3-epi-oleanolic acid are not clear.

Disclosure of Invention

The present inventors have found, through intensive studies, that 3-epioleanolic acid can inhibit the binding of the S protein of neocoronavirus and ACE2, and thus can be used for preventing and/or treating infection with the neocoronavirus, and have completed the present application based on the finding.

A first aspect of the application provides the use of 3-epioleanolic acid in the manufacture of a medicament for the prevention and/or treatment of new coronavirus infections.

A second aspect of the present application provides a pharmaceutical composition for preventing and/or treating a new coronavirus infection, comprising 3-epioleanolic acid.

The 3-epioleanolic acid can inhibit the combination of the S protein of the new coronavirus and ACE2, so that the 3-epioleanolic acid can be used for preventing and/or treating the infection of the new coronavirus, and further can be used for preparing a medicine for preventing and/or treating the infection of the new coronavirus. Further, the pharmaceutical composition comprising 3-epioleanolic acid can also be used for preparing a medicament for preventing and/or treating new coronavirus infection.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present application, and it is also obvious for a person skilled in the art to obtain other embodiments according to the drawings.

FIG. 1 shows the results of the inhibition of the binding of S protein to ACE2 receptor by 3-epi-oleanolic acid, wherein A is the inhibition result with the concentration of 3-epi-oleanolic acid as abscissa; the graph B shows the inhibition results with lg (concentration of 3-epi-oleanolic acid) as the abscissa.

FIG. 2 shows the results of 3-epi-oleanolic acid inhibiting pseudovirus infection of Opti-HEK293/ACE2 cells, wherein A is the results of luciferase activity of 3-epi-oleanolic acid inhibiting pseudovirus infection of Opti-HEK293/ACE2 cells, B is the results of inhibition rate of 3-epi-oleanolic acid inhibiting pseudovirus infection of Opti-HEK293/ACE2 cells with lg (concentration of 3-epi-oleanolic acid) as abscissa.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the description herein are intended to be within the scope of the present disclosure.

A first aspect of the application provides the use of 3-epioleanolic acid in the manufacture of a medicament for the prevention and/or treatment of new coronavirus infections.

The inventor finds in research that the 3-epioleanolic acid can inhibit the binding of the S protein of the novel coronavirus and ACE2, so that the 3-epioleanolic acid can be used for preventing and/or treating the infection of the novel coronavirus, and further can be used for preparing a medicament for preventing and/or treating the infection of the novel coronavirus.

The term "treatment" has its ordinary meaning in the present application and refers herein in particular to the treatment of a mammalian subject (preferably a human) already suffering from a new coronavirus infection with a medicament according to the present application in order to produce a therapeutic, curative, palliative etc. effect on said disease. Similarly, the term "prevention" as used herein has its ordinary meaning and refers herein in particular to the treatment of a mammalian subject who may be suffering from or at risk of suffering from a new coronavirus infection with a medicament of the present application in order to produce a preventing, arresting, abrogating, etc. effect on said disease.

In some embodiments of the first aspect of the present application, the new coronavirus infection comprises at least one of pneumonia and acute respiratory distress syndrome caused by the new coronavirus infection.

In some embodiments of the first aspect of the present application, 3-epioleanolic acid prevents and/or treats a new coronavirus infection by inhibiting the binding of the new coronavirus S protein to host ACE 2.

A second aspect of the present application provides a pharmaceutical composition for preventing and/or treating a new coronavirus infection, comprising 3-epioleanolic acid.

In some embodiments of the second aspect of the present application, the 3-epi oleanolic acid is provided in the form of a monomer, or in the form of a plant extract comprising the same.

In some embodiments of the second aspect of the present application, the plant extract is selected from at least one of a hance berry leaf extract, a barbed skullcap herb extract and a verbena extract.

The extraction methods of the Chinese mahogany leaf extract, the sculellaria barbata extract and the verbena extract are reported in the prior art, the extraction method of each medicine extract is not particularly limited in the application, and a person skilled in the art can obtain the Chinese mahogany leaf extract, the sculellaria barbata extract or the verbena extract containing 3-epioleanolic acid in any conventional manner. In one embodiment, verbena may be subjected to heat reflux extraction with 70-80% ethanol solution, to obtain verbena extract containing 3-epi-oleanolic acid, for example.

In some embodiments of the second aspect of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

Herein, "pharmaceutically acceptable" means having no substantial toxic effect when used in the usual dosage amounts, and thus being approved by the government or equivalent international organization or approved for use in animals, more particularly in humans, or registered in the pharmacopoeia.

The "pharmaceutically acceptable carrier or excipient" useful in the pharmaceutical compositions of the present application may be any conventional carrier in the art of pharmaceutical formulation, and the selection of a particular carrier will depend on the mode of administration or the type and state of the disease used to treat a particular patient. The preparation of suitable pharmaceutical compositions for a particular mode of administration is well within the knowledge of those skilled in the pharmaceutical art. For example, solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, mold release agents, coating agents, flavors, antioxidants, and the like, which are conventional in the pharmaceutical field, may be included as the pharmaceutically acceptable carrier or excipient. If necessary, a flavor, a preservative, a sweetener and the like may be further added to the pharmaceutical composition.

As used herein, the term "pharmaceutical composition" has its ordinary meaning. In addition, the "pharmaceutical composition" of the present application may also be present or provided in the form of a health product, a functional food, a food additive, or the like. The pharmaceutical compositions of the present application can be prepared by conventional techniques in the pharmaceutical field, particularly in the formulation field, by obtaining the active ingredients of the raw materials of the pharmaceutical compositions of the present application by extraction, separation and purification means commonly used in pharmaceutical manufacturing, optionally mixing with one or more pharmaceutically acceptable carriers or excipients, and then forming the desired dosage form. The pharmaceutical composition according to the present application is a pharmaceutical preparation which can be suitably used for oral administration, a pharmaceutical preparation (e.g., solution) suitable for parenteral injection (e.g., intravenous injection, subcutaneous injection), a pharmaceutical preparation (e.g., ointment, patch or cream) suitable for surface administration, or a pharmaceutical preparation (e.g., suppository) suitable for rectal administration, and the like. Dosage forms for oral administration may include, for example, tablets, pills, drop pills, hard or soft capsules, solutions, suspensions, emulsions, tinctures, syrups, powders, fine granules, pellets, elixirs and the like, without limitation. In addition to the active ingredient, these preparations may contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), lubricants (e.g., silica, talc, stearic acid or its magnesium salt, calcium salt, and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone. If necessary, it may further contain pharmaceutically acceptable additives such as disintegrating agents (e.g., starch, agar, alginic acid or sodium salt thereof), absorbents, coloring agents, flavoring agents, sweetening agents, and the like. Tablets may be prepared according to conventional mixing, granulating or coating methods.

The pharmaceutically acceptable dose of the pharmaceutical composition of the present application, i.e., the administration dose, in terms of 3-epioleanolic acid, may vary according to the age, sex and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the administration route and the judgment of the diagnostician. Determining the dosage to be administered taking these factors into account is within the level of skill in the art. A typical dose may be 0.01-1000 mg/kg/day, specifically 1-100 mg/kg/day. However, the scope of the present disclosure is not in any way limited by the administration dosage.

The present application will be described in detail with reference to specific examples.

3-Epiooleanolic acid (3-Epiolelanolic acid, CAS number: 25499-90-5), available from Shanghai-derived leaf Biotech, Inc., having the following structural formula:

the experimental materials and methods used in the following examples are, unless otherwise specified, conventional materials and methods.

Example 1 molecular docking method to predict the interaction of 3-epi-oleanolic acid with S protein, ACE2 receptor

Preparation of small molecule ligand 3-epi oleanolic acid: downloading the 3D structure of 3-epi-oleanolic acid from http:// www.chemspider.com/; and (3) completing the structure: adopting Discovery Studio2020 software, expanding Small Molecules/Prepare or Filter Ligands in a toolbar, clicking the Prepare Ligands, generating a three-dimensional structure through the operation, and hydrogenating to generate isomers and the like; optimizing the structure: expand Small Molecules/Minimize Ligands in toolbar, click on Full Minimization (parameters typically use defaults); and (5) storing the optimized small molecular structure.

Preparation of macromolecular receptor: the 3D structure of the S protein-ACE 2 receptor complex was downloaded from a protein database (http:// www.rcsb.org /), and the PDB accession number: 6LZG (resolution: 2.50A °); and (3) macromolecular optimization treatment: opening Discovery Studio2020 software, expanding Macromolecules/Prepare Protein/Manual Preparation in a tool bar, clicking clear Protein, expanding Macromolecules/Prepare Protein/Automatic Preparation in the tool bar, clicking the Prepare Protein, deleting water molecules and heteroatoms, hydrogenating, dehydrating, completing a structure, setting a protonation state and the like; the optimized receptor structure is stored as a "6 LZG + prep" file.

Definition of the active center of the receptor: it has been reported that the S protein of SARS-CoV-2 virus binds to human ACE2 receptor, thereby causing the virus to invade the human body. In this example, the protein-protein interaction (PPI) between the S protein and ACE2 in the S protein-ACE 2 receptor complex was first analyzed, and the sites (sites) that may disrupt the binding of the S protein to ACE2 were identified; as shown in Table 1, the action sites on the two binding interfaces can be divided into three regions, the three regions comprise six binding sites, the active centers are respectively defined according to the amino acid positions of the different binding sites, namely S-site1, S-site2, S-site3, ACE2-site1, ACE2-site2 and ACE2-site3, and the receptor structures after the active centers are defined are additionally stored as 6LZG + prep + active files.

TABLE 1

The active site of binding of the S protein-ACE 2 receptor complex to each other was determined according to table 1: according to the interaction between S protein and ACE2 receptor, the binding Site can be divided into three regions, namely Site1, Site2 and Site 3; the Site1 region consists of the interaction of TYR449, GLY496, GLN498, THR500, ASN501, GLY502, TYR505 residues on the S protein with GLN42, LYS353, GLY354, ASP355 residues on the ACE2 receptor surface; the Site2 region consists of LYS417, GLN493, LEU455 residues on the S protein interacting with ASP30 and HIS34 residues on the surface of ACE2 receptor; the Site3 region consists of the interaction of GLU484, PHE486, ASN487, TYR489 residues on the S protein with GLN24, PHE28, LYS31, MET82, TYR83 residues on the surface of the ACE2 receptor.

Performing CDOCKER molecular docking on the optimized structure of the 3-epi-oleanolic acid and an active center defined by an S protein-ACE 2 complex respectively; storing and analyzing the 2D graph of the docking result; using the binding ENERGY (Interactive ENERGY) as a score, generally, the lower the value of the binding ENERGY, the stronger the binding affinity of the compound to form a stable complex with S protein-ACE 2, and thus, the stronger the binding inhibition of S protein-ACE 2 was shown. The binding energy results of 3-epi-oleanolic acid with ACE2 and S protein are shown in Table 2, and the results show that 3-epi-oleanolic acid can be respectively bound with three sites of ACE2, wherein the binding with site2 is better, and the value of the binding energy is-29.0597 kJ/mol; 3-oleanolic acid can be respectively combined with two sites of the S protein, wherein the combination with site1 is better, and the combination energy value is-26.4275 kJ/mol.

TABLE 2

The amino acid residues of the 3-epi-oleanolic acid, SARS-CoV-2 surface S protein and ACE2 receptor are shown in Table 3; wherein 3-epi oleanolic acid forms hydrogen bond and charge interaction with ACE2 protein residue LYS353 at site1, and forms Alkyl and Pi-Alkyl hydrophobic interaction with residue TYR 41; and hydrogen bonds are formed with S protein residues ASN501 and THR500 respectively. Hydrogen bonds and charge interaction are formed between site2 and ACE2 protein residue LYS31, and Pi-Alkyl hydrophobic interaction is formed between site2 and residue HIS 34; forms an Alkyl and Pi-Alkyl hydrophobic interaction with S protein residue ARG403, and forms a charge interaction with residues LYS417, TYR453 and LEU455 respectively. Hydrogen bonds and Pi-Alkyl hydrophobic interaction are respectively formed between site3 and ACE2 protein residues MET82, TYR83 and ILE 21; does not bind to the S protein and has no interaction. The results indicate that 3-epi oleanolic acid can bind to the S protein site, ACE2 receptor site.

TABLE 3

Example 23 Effect of Epoleanolic acid on binding of S protein to ACE2 receptor

The detection kit for the SARS-CoV-2 neutralizing antibody has the following principle: the kit is a neutralizing antibody blocking ELISA (enzyme linked immunosorbent assay) detection tool, and comprises two key components: HRP (horse radish peroxide) labeled recombinant SARS-CoV-2RBD fragment and human ACE2 receptor protein (hACE 2); the combination of the RBD antigen of the S protein binding domain of the new coronavirus and the ACE2 antigen of the receptor protein is utilized to simulate the interaction between the virus and host cells, and the virus infection condition is judged through a chromogenic reaction, but when a neutralizing antibody exists, the interaction between the S protein and the ACE2 receptor is blocked, and the chromogenic reaction is weakened.

1) Dissolution of monomer components: dissolving 3-epi oleanolic acid in dimethyl sulfoxide (DMSO, Beijing Solebao technologies Co., Ltd.) to prepare monomer solutions with concentrations of 100mM (mmol/L), 80mM, 60mM, 50mM and 30mM respectively; 2) using a SARS-CoV-2 neutralizing antibody detection kit (Cat.No.: L00847-A, Nanjing Kingsry Biotech Co., Ltd.), the temperature was returned to room temperature before use; 3) diluting a 3-epi-oleanolic acid monomer solution, a positive control and a negative control in a reagent kit by using a sample diluent in the reagent kit according to a ratio of 1:9, wherein the concentrations of the diluted 3-epi-oleanolic acid are 10mM, 8mM, 6mM, 5mM and 3mM respectively; 4) mixing the diluted monomer solution, negative control and positive control with recombinant SARS-CoV-2RBD fragment (HRP-RBD) labeled by horseradish peroxide according to volume ratio of 1:1 to prepare 100 μ L of each compound mixed solution, and incubating at 37 deg.C for 30 min; 5) respectively adding 100 mu L of each compound mixed solution into an enzyme label plate coated with ACE2 to obtain each monomer administration group, a negative control group and a positive control group, covering the plate with a cover plate film, and then incubating for 15 minutes at 37 ℃; wherein the final concentrations of the 3-epioleanolic acid in each monomer administration group are 5mM, 4mM, 3mM, 2.5mM and 1.5mM respectively; 6) the cover plate membrane was removed and the plate washed 4 times with 260. mu.L of 1 Xwash solution; 7) adding 100 mu L of TMB Solution into each hole, and incubating for 15 minutes at 20-25 ℃ in the dark; 8) adding 50 mu L of stop solution into each hole to stop the reaction; 9) immediately after termination, the samples were analyzed by microplate reader (diken (shanghai) trade ltd, model: INFINITEF50) at 450 nm.

According to the absorbance value, by the formula: the inhibition rate is 1-absorbance value of monomer administration group/absorbance value of negative control group multiplied by 100%, and the inhibition rate of 3-epi-oleanolic acid on the combination of S protein and ACE2 receptor is calculated; wherein, the concentration (mM) of 3-epi-oleanolic acid is used as the abscissa, and the inhibition rate result is shown in A diagram of figure 1; the results of inhibition ratios are shown in B chart of FIG. 1 with lg (concentration of 3-epi-oleanolic acid) as the abscissa; the results of panels A and B in FIG. 1 show that 3-epi-oleanolic acid can inhibit the binding of S protein of SARS-CoV-2 and ACE2 receptor in a concentration-dependent manner, wherein the inhibition rate at 5mM is 66.96%; further calculation of the semi-Inhibitory Concentration (IC)₅₀) Numerical values, results show the IC of 3-epi-oleanolic acid₅₀The value was 3.633 mM. The results show that 3-epi-oleanolic acid inhibits the binding of the S protein to ACE2 receptor in a concentration-dependent manner, and 3-epi-oleanolic acid inhibitsIC for binding of S-producing protein to ACE2 receptor₅₀The value was 3.633 mmol/L.

Example 33 Effect of Epiglycemic acid on infection of Opti-HEK293/ACE2 cells by pseudoviruses

The principle of the pseudovirus neutralizing antibody detection kit is as follows: the envelope glycoprotein in the lentiviral vector is replaced by the new coronavirus S protein to form a pseudovirus simulating the infection of the new coronavirus; the pseudovirus infects target cells through surface S protein and expresses a reporter luciferase gene, and the blocking degree of the virus can be deduced by detecting the expression quantity of the reporter gene luciferase, so that the neutralizing agent is screened or verified; neutralizing agents (e.g., antibodies) can block the binding of the S protein to ACE2, thereby preventing infection of the host cell by the pseudovirus.

The SARS-CoV-2 pseudovirus neutralization detection kit (Nanjing Kingsry Biotechnology Co., Ltd.) was used: 1) preparation of monomer components: dissolving 3-epioleanolic acid in DMSO, and preparing monomer solution with concentration of 160 μ M, 112 μ M, 40 μ M, 10 μ M, 2.8 μ M in Opti-MEM culture medium; 2) preparation of positive antibody: adding 5 mu L of 1 mu g/mu L positive antibody into 120 mu L LOpti-MEM culture medium to prepare a positive antibody solution of 40 mu g/mL; 3) taking pseudovirus (from the kit), placing the pseudovirus in a water bath at 37 ℃ for rapid re-melting, adding the virus into a 15ml centrifuge tube containing 1500 mu L of Opti-MEM culture medium, and uniformly mixing to obtain a pseudovirus solution; 4) in each monomer administration group, monomer solution with each concentration is added into each group at 25 mu L/hole; adding a positive antibody solution into the positive control group at a rate of 25 mu L/hole; adding 25 μ L of Opti-MEM culture medium into the negative control group and the blank control group respectively; adding 25 mu L/hole pseudovirus solution into the monomer administration group, the positive control group and the negative control group respectively, and adding 25 mu L/hole Opti-MEM culture medium into the blank control group; wherein the final concentrations of 3-epi-oleanolic acid in each monomer administration group are respectively 80 μ M, 56 μ M, 20 μ M, 5 μ M and 1.4 μ M; 5) respectively mixing with pseudovirus solution, and incubating at room temperature for 1 h; 6) placing an Opti-HEK293/ACE2 cell (derived from the kit, namely an HEK293 cell over expressing ACE2) in a water bath at 37 ℃ to rapidly re-fuse the cell; 7) transferring the cells to a 15ml centrifuge tube, adding 4ml of preheated DMEM complete culture medium, centrifuging at 100 Xg for 5min, and discarding the supernatant; 8) suspending the cells in 4ml of pre-warmed DMEM complete medium, fully mixing, counting the cells and adjusting the density of the cell suspension to 600000 cells/ml; 9) after the incubation in the step 5) is finished, adding 50 mu l of the cell suspension in the step 8) into each hole; after the edge hole of the 96-well plate is sealed by PBS, putting the 96-well plate into a cell culture box; 10) adding 50 mu L of preheated fresh DMEM complete culture medium into each hole after 24 hours, and continuously putting the mixture into an incubator for 24 hours; 11) sealing the 96-hole cell culture microporous plate with the white wall and the transparent bottom by using a microporous plate sealing film; carefully absorbing and discarding the culture medium of the 96-well plate, immediately adding 50 mul of newly-prepared luciferase developing solution, and incubating for 3-5 minutes at room temperature; 12) the microplate luminescence detector detects the chemiluminescence value, and the signal value is read, namely the luciferase activity result.

The data obtained were statistically analyzed using the software GraphPad Prism 8.0.1 and Excel, all values being expressed as mean ± SD, and statistical comparisons were performed using paired t-tests, one-way analysis of variance (ANOVA). P is less than 0.05, which is significant.

The luciferase activity of each group was counted, and the results are shown in a graph a of fig. 2, and 3-epi oleanolic acid was able to reduce the luciferase activity of Opti-HEK293 cells after overexpression of ACE2 (final concentrations were 56 μ M and 80 μ M, respectively) compared to the negative control group, indicating that 3-epi oleanolic acid can effectively prevent entry of pseudovirus into the cells (n ═ 3, × P < 0.001, compared to the negative control group); from the read signal values, by the formula: the inhibition rate was 1- (signal value of monomer administered group-signal value of blank control group)/(signal value of negative control group-signal value of blank control group) × 100%, the inhibition rate of 3-epi oleanolic acid against pseudovirus-infected cells was calculated, lg (concentration of 3-epi oleanolic acid) was used as abscissa, the inhibition rate results were shown in graph B of fig. 2, and IC was further calculated₅₀Numerical value, result display IC₅₀The value was 45.71. mu.M. The results show that 3-epi oleanolic acid can inhibit the pseudovirus containing the S protein of the new coronavirus from infecting Opti-HEK293/ACE2 cells, and the 3-epi oleanolic acid can inhibit the IC of the pseudovirus from infecting Opti-HEK293/ACE2 cells₅₀The value was 45.71. mu. mol/L.

In conclusion, the 3-epi oleanolic acid can inhibit the combination of the new coronavirus S protein and ACE2 and inhibit pseudovirus infected cells containing the new coronavirus S protein, so that the 3-epi oleanolic acid can inhibit the combination of the new coronavirus S protein and host ACE2, can be used for preventing and/or treating the new coronavirus infection, and can be further used for preparing the medicine for preventing and/or treating the new coronavirus infection.

The above description is only for the preferred embodiment of the present application and is not intended to limit the scope of the present application. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application are included in the protection scope of the present application.