Use of cepharanthine in the preparation of a medicament for inhibiting SADS-CoV

Abstract

The present invention relates to the use of cepharanthine or its prodrug or its salt in the preparation of an inhibitor of swine acute diarrhea syndrome coronavirus (SADS-CoV), and the use of cepharanthine or its prodrug or its salt in the preparation of a drug for preventing and / or treating SADS-CoV infectious diseases. The present invention has confirmed through experiments that cepharanthine can significantly inhibit the infection of SADS-CoV virus on Huh-7 cells, and it can play an obvious inhibitory role at the stage of virus entry into cells, and it is a potential antiviral drug for treating SADS-CoV infectious diseases.

Description

[0001] Cross-reference

[0002] This application claims the priority of a Chinese patent application with the application number 202210568741.4 and the invention title "Use of cepharanthine in the preparation of a drug for inhibiting SADS-CoV" filed on May 24, 2022, the entire content of which is incorporated herein by reference. Technical field

[0003] The present invention belongs to the field of biomedicine and relates to a new use of a known drug. Specifically, it relates to the use of cepharanthine or its prodrug or its salt in the preparation of a drug for inhibiting swine acute diarrhea syndrome coronavirus (SADS-CoV). Background art

[0004] Coronaviruses are a general term for a class of single-stranded positive-strand RNA viruses, belonging to the order Nidovirales, family Coronaviridae, genus Coronavirus, and can be divided into four genera: α, β, γ, and δ. The genome size of coronaviruses ranges from 26 to 32 kb, and mainly encodes structural proteins including spike protein (S protein), envelope protein (E protein), membrane protein (M protein), and nucleocapsid protein (N protein), as well as other non-structural proteins (nsp) encoded by the functional region ORF1ab. Among them, coronaviruses usually enter cells by binding to surface receptors through the S protein.

[0005] Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an α-coronavirus that can cause fatal swine acute diarrhea syndrome and spread in Guangdong and Fujian regions from 2017 to 2019. SADS-CoV infection can cause severe acute vomiting and diarrhea in neonatal piglets, a sharp drop in body weight, and cause large-scale death of piglets in a short time, bringing huge economic losses and negative impacts to the pig farming industry and related industries.

[0006] SADS-CoV has a sequence homology of more than 95% with bat coronavirus HKU2, and the two belong to the same genus of α-coronavirus in taxonomy. The spike proteins of SADS-CoV (1130 amino acid residues) and HKU2 (1128 amino acid residues) are among the shortest spike proteins of coronaviruses, and their amino acid sequence homologies with other known coronavirus spike proteins are relatively low, indicating the particularity of HKU2 and SADS-CoV in evolution.

[0007] In addition, some studies have shown that as a highly pathogenic pathogen, SADS-CoV has the potential for cross-species transmission and may pose a threat to human life and health in the future.

[0008] Currently, there are no available vaccines and antiviral drugs for SADS-CoV infection, and the number of related studies is limited. In the face of possible future SADS-CoV transmission, the research and development of antiviral drugs against SADS-CoV become very crucial.

[0009] Cepharanthine is a bisbenzylisoquinoline alkaloid isolated from the rhizomes of Stephania cepharantha Hayata. Cepharanthine has anti-inflammatory, antibacterial, and immunomodulatory functions. In recent years, many scholars believe that it also has functions such as stimulating the reticuloendothelial system, activating hematopoietic tissues, and promoting the proliferation of bone marrow tissues. Clinically, it can be used to prevent and treat leukopenia caused by radiotherapy or chemotherapy in cancer patients, and it has achieved remarkable results. Summary of the Invention

[0010] Object of the Invention

[0011] The purpose of the present invention is to provide the use of cepharanthine or its prodrug or its salt in the preparation of a porcine acute diarrhea syndrome coronavirus inhibitor, and to provide the use of cepharanthine or its prodrug or its salt in the preparation of a drug for preventing and / or treating porcine acute diarrhea syndrome coronavirus infectious diseases.

[0012] Solution

[0013] To achieve the above purpose, the present invention provides the following technical solutions:

[0014] In the first aspect, the present invention provides the use of cepharanthine or its prodrug or its salt in the preparation of a porcine acute diarrhea syndrome coronavirus inhibitor.

[0015] In the second aspect, the present invention provides the use of cepharanthine or its prodrug or its salt in the preparation of a drug for preventing and / or treating porcine acute diarrhea syndrome coronavirus infectious diseases.

[0016] In the above uses, the prevention and / or treatment of porcine acute diarrhea syndrome coronavirus infectious diseases is to inhibit the activity of porcine acute diarrhea syndrome coronavirus.

[0017] Further, the inhibition of the activity of porcine acute diarrhea syndrome coronavirus includes: inhibiting the replication of porcine acute diarrhea syndrome coronavirus, and / or inhibiting the entry of porcine acute diarrhea syndrome coronavirus into cells.

[0018] In the above uses, the prodrug of cepharanthine refers to a drug that can be converted into cepharanthine in vivo.

[0019] In the above uses, preferably, the salt of cepharanthine is cepharanthine hydrochloride, cepharanthine sulfate, cepharanthine acetate or cepharanthine sulfonate.

[0020] In the above uses, preferably, the dosage form of the drug is injection, oral liquid, powder, tablet, granule, capsule, syrup, powder for injection, aqueous injection, decoction, medicinal liquor, sustained-release preparation, enteric-coated preparation, aerosol or suspension.

[0021] Beneficial Effects

[0022] The inventor of the present application used human hepatoma cell Huh-7 as a model and screened out an active compound, cepharanthine, which can significantly inhibit the activity of SADS-CoV and thus can be used for the prevention and / or treatment of SADS-CoV infectious diseases. The present invention confirmed through a series of experiments that cepharanthine or its prodrug or its salt can significantly inhibit the infection of Huh-7 cells by SADS-CoV virus, and this inhibitory effect is significantly dose-dependent. Specifically, cepharanthine or its prodrug or its salt has an obvious inhibitory effect on the infection of Huh-7 cells by SADS-CoV virus and has low drug cytotoxicity (its EC 50 = 1.546 μM, CC 50 = 24.61 μM, and the SI value is 15.92), and it was found that an obvious inhibitory effect can be achieved at the stage of virus entry into cells. Therefore, it can be used as a potential candidate drug for the treatment of SADS-CoV infectious diseases. Description of the Drawings

[0023] One or more embodiments are exemplarily illustrated by the pictures in the corresponding drawings. These exemplary illustrations do not constitute a limitation on the embodiments. Here, the special word "exemplary" means "serving as an example, embodiment or illustration". Here, any embodiment illustrated as "exemplary" does not have to be construed as superior to or better than other embodiments.

[0024] Figure 1 Showing the cytopathic effects of different concentrations of cepharanthine inhibiting SADS-CoV infection. Among them, A and B are the virus infection conditions after adding 6.25 μM and 3.125 μM cepharanthine respectively; C is the virus addition control; D and E are the virus infection conditions after adding 1.5625 μM and 0.78125 μM cepharanthine respectively; F is the blank control.

[0025] Figure 2It shows the dose-dependent change of tetrandrine at different concentrations in inhibiting the infection of SADS-CoV to Huh-7 cells, as well as the cytotoxicity change of tetrandrine at different concentrations to Huh-7 cells.

[0026] Figure 3 It shows a schematic diagram of the experimental procedure of the drug addition time experiment described in Example 4.

[0027] Figure 4 It shows the content of viral RNA in cells 48 hpi after adding 6.25 μM of tetrandrine at different stages of virus infection, where DMSO represents the virus-added control; Entry, Post entry, and Full time respectively represent drug addition treatments before virus entry, after virus entry, and throughout the virus infection process.

[0028] Figure 5 It shows the cell morphology diagrams 48 hours after adding 6.25 μM of tetrandrine at different stages of virus infection. Among them, A is the Entry group treated before virus entry, B is the Post entry group treated after virus entry, D is the Full time group treated with the drug throughout the virus infection process, and C and E are the virus-added control group and the blank control group respectively.

[0029] Figure 6 It shows the result diagrams of plaque assays using the supernatants of different dilutions of the virus-added control group in the drug addition time experiment.

[0030] Figure 7 It shows the result diagrams of plaque assays using the supernatants of different dilutions of the group treated with the drug before virus entry (Entry) in the drug addition time experiment.

[0031] Figure 8 It shows the result diagrams of plaque assays using the supernatants of different dilutions of the group treated with the drug after virus entry (Post entry) in the drug addition time experiment.

[0032] Figure 9 It shows the result diagrams of plaque assays using the supernatants of different dilutions of the group treated with the drug throughout the virus infection process (Full time) in the drug addition time experiment.

[0033] Figure 10 It shows that in the virus adsorption experiment, the content of viral RNA adsorbed on cells in each experimental group was detected by qPCR method; among them, the abscissa shows the experimental group, and the ordinate shows the relative expression level of viral RNA.

[0034] Figure 11Shows the virus titer in cells after 24 hpi after virus infection following co-incubation of 6.25 μM cepharanthine with cells, with virus, or with both cells and virus in a drug incubation experiment; wherein, the abscissa shows the experimental groups and the ordinate shows the virus titer (i.e., TCID 50 / mL) level. Detailed implementation manners

[0035] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the protection scope of the present invention. Unless otherwise clearly stated, in the whole specification and claims, the term "comprise" or its variations such as "comprises" or "comprising" will be understood to include the stated elements or components, without excluding other elements or other components.

[0036] In addition, for better illustration of the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In some embodiments, raw materials, components, methods, means, etc. well-known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

[0037] The reagents, reagent kits, raw materials and equipment, etc. used in the present invention can be obtained through commercial channels without special instructions. The experiments or detection methods involved in the present invention are conventional experiments or detection methods in the art without special instructions, or are carried out with reference to the corresponding reagent kits or product specifications.

[0038] Hereinafter, the embodiments of the present invention will be described in detail.

[0039] General Experimental Method

[0040] Cell and Virus Cultivation Method

[0041] The human hepatoma cell line Huh-7 used in the following examples was obtained from the American Type Culture Collection (ATCC); it was cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; PAN) under the conditions of 37 °C and 5% CO2.

[0042] The virus used in the following examples is the isolate SADS-CoV / CN / GDWT / 2017 (Genbank accession number: MG557844); this SADS-CoV virus strain was used to infect Huh-7 cells at an MOI of 0.1 as a virus infection model to explore the antiviral effect of cepharanthine against SADS-CoV infection; the virus titer was determined using the plaque assay.

[0043] All virus infection experiments were conducted in a Biosafety Level 2 (BLS2) laboratory.

[0044] Virus RNA Extraction and Quantitative Real-time PCR (qPCR)

[0045] Huh-7 cells after virus infection were collected, and virus RNA was extracted using a Tissue / Cell Total RNA Extraction Kit (Beijing Nobel Biotechnology Co., Ltd., product number RNE11-02) according to the manufacturer's instructions.

[0046] Using Ⅱ 1st Strand cDNA Synthesis SuperMix for qPCR (Shanghai Yisheng Biotechnology Co., Ltd., product number 11123ES60), the extracted virus RNA was reverse-transcribed; and, using the reverse-transcribed cDNA as a template, qPCR SYBR Green Master Mix (Shanghai Yisheng Biotechnology Co., Ltd., product number 11202ES08) and QuantStudio TM 1 Real-Time Fluorescence Quantitative PCR System (Applied-Biosystem) was used for qPCR detection; the sequence information of the primers used is shown in Table 1. The detection method for qPCR amplification was the SYBR-Green dye method, and the amplification program was 95°C for 5 min; 95°C for 10 s, 55°C for 20 s, 72°C for 31 s, for 40 cycles. GraphPad-Prism 8.0.2 software was used to analyze the data results and create graphs.

[0047] Table 1 Primer sequences used in the study

[0048]

[0049] Drug Source

[0050] Cepharanthine powder (product number T0131) was purchased from Shanghai Tauto Biochemical Technology Co., Ltd. and dissolved in dimethyl sulfoxide (DMSO) to a stock concentration, such as 10 mM; then, when used, it was first diluted to 1 mM with phosphate-buffered saline (PBS) and then further diluted to the required working concentration with the medium.

[0051] Example 1: Inhibitory effect of cepharanthine on SADS-CoV virus-infected Huh-7 cells

[0052] To detect the inhibitory effect of cepharanthine on SADS-CoV-infected Huh-7 cells, the inventor seeded Huh-7 cells at a density of 2×10 4 cells per well in a 96-well plate and performed the experiment 18 - 24 hours later. The cepharanthine powder was dissolved in dimethyl sulfoxide (DMSO) to 10 mM, then diluted to 1 mM with PBS, and further diluted to 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM, 0.390625 μM with culture medium. The virus dilution was mixed with the above series of drug dilutions at a ratio of 1:1 and added to the cells, finally resulting in a virus infection multiplicity of infection (MOI) = 0.1, and the final drug concentrations were 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM, 0.390625 μM, 0.1953125 μM. In addition, for the virus-added control group, the culture medium containing 0.1% DMSO was mixed with the virus dilution at a ratio of 1:1 and added to the cells, with a final virus infection MOI = 0.1; for the blank control group, the culture medium containing 0.1% was mixed with the culture medium without drug or virus at a ratio of 1:1 and added to the cells. The cells were incubated at 37 °C and 5% CO2. After 2 hours of incubation, the supernatant was removed, the cells were washed with phosphate-buffered saline (PBS; HyClone), and then the culture medium containing the same drug concentration was added, and the cells were continued to be cultured until 48 hours. When cytopathic effects appeared, the cytopathic conditions of cells in each drug concentration group were observed using an electron microscope.

[0053] The cytopathic conditions of the cells treated with 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM cepharanthine are shown in Figure 1 A, 1B, 1D, 1E respectively, and the virus-added control and blank control are shown in Figure 1 C and Figure 1 F respectively; It can be seen from Figure 1 that compared with the virus-added control, the number of cells with cytopathic death in the experimental group with cepharanthine added decreased with the increase of drug concentration. Therefore, it is preliminarily speculated that cepharanthine can effectively inhibit the infection of SADS-CoV to Huh-7 cells.

[0054] Example 2: Detection of inhibitory activity of cepharanthine on SADS-CoV virus infection

[0055] In this example, the inhibitory effect of cepharanthine at different concentrations on SADS-CoV infection in Huh-7 cells was detected and the EC50 Value, the specific procedure is as follows:

[0056] Inoculate Huh-7 cells into 96-well plates at a density of 2×10 4 cells per well and perform the experiment 18 - 24 hours later. Dissolve tetrandrine powder in dimethyl sulfoxide (DMSO) to 10 mM, then dilute it to 1 mM with PBS, and then further dilute it to 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM, 0.390625 μM with culture medium respectively. Mix the virus dilution with the above series of drug dilutions at a ratio of 1:1 and add them to the cells, finally making the multiplicity of infection (MOI) of virus infection = 0.1, and the final concentrations of the drugs are 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM, 0.390625 μM, 0.1953125 μM respectively. In addition, for the virus-added control group, the treatment is to mix the culture medium containing 0.1% DMSO with the virus dilution at a ratio of 1:1 and add it to the cells, finally making the MOI of virus infection = 0.1; for the blank control group, the treatment is to mix the culture medium containing 0.1% with the culture medium without drugs or viruses at a ratio of 1:1 and add it to the cells. Incubate the cells under the conditions of 37 °C and 5% CO2. After incubating for 2 hours, remove the supernatant, wash it with phosphate-buffered saline (PBS; HyClone), add the culture medium containing the same drug concentration, and continue to culture until 48 hours when cytopathic effect appears. Then collect the cell nucleic acid and perform qPCR detection to determine the virus RNA content in the cells, and further calculate the inhibition rate (%) of drugs at different concentrations on SADS-CoV infection. Use GraphPad-Prism 8.0.2 software to analyze the data results. Take the tetrandrine concentration as the abscissa and the virus inhibition rate (%) as the ordinate to make a virus inhibition rate (%) vs drug concentration curve, and fit this curve to obtain the EC 50 value. The calculation formula for the inhibition rate (%) is as follows:

[0057]

[0058] The virus inhibition rates at different concentrations of tetrandrine are as Figure 2 (left y-axis) shown; Figure 2 It also shows the EC 50 of tetrandrine in inhibiting SADS-CoV infection, which is 1.546 μM. This experiment was repeated 3 times, and the results of the 3 experiments were consistent or approximate.

[0059] By Figure 2From the virus inhibition rate (%) vs drug concentration curve, it can be seen that the inhibitory effect of cepharanthine on SADS-CoV-infected Huh-7 cells shows a dose-dependence, that is, within the range of drug toxicity, as the concentration of cepharanthine increases, the inhibition rate of virus infection gradually increases, and the inhibitory effect also gradually increases; at the same time, it can be seen that cepharanthine can inhibit 50% of cell virus infection at a concentration of 1.546 μM. Therefore, the results show that cepharanthine can significantly inhibit SADS-CoV infection of Huh-7 cells.

[0060] Example 3: Cytotoxicity detection of cepharanthine

[0061] In this example, the cytotoxicity of cepharanthine at different concentrations on Huh-7 cells was detected, and the CC 50 value was estimated. The specific procedure is as follows:

[0062] The CellTiter-Blue method was used to detect cell viability. The specific method is as follows: Huh-7 cells were seeded in 96-well plates at a density of 2×10 4 cells per well and incubated for 18 - 24 hours before the experiment. Cepharanthine powder was dissolved in dimethyl sulfoxide (DMSO) to 10 mM, then diluted to 1 mM with PBS, and then further diluted to 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM, 0.390625 μM, 0.1953125 μM with culture medium. The diluted drugs were added to the cells and incubated at 37 °C and 5% CO2 for 48 hours. Then, 20 μL of resazurin (CellTiter-Blue; Promega) was added to each well, and the luminescence intensity of each well at 593 nm was measured using a microplate reader at 0 min, 30 min, 60 min, 90 min, and 120 min. GraphPad-Prism 8.0.2 software was used to analyze the data results and calculate the cytotoxicity (%). And, with the cepharanthine concentration as the abscissa and the cytotoxicity (%) as the ordinate, a cytotoxicity (%) vs drug concentration curve was made, and the CC 50 value was obtained by fitting this curve. The cytotoxicity (%) was calculated by detecting the effect of cepharanthine at different concentrations on cell viability (the detected fluorescence intensity can reflect cell viability), and its calculation formula is as follows:

[0063]

[0064] The cytotoxicity percentages at different cepharanthine concentrations are as Figure 2 (right y-axis) shown; Figure 2 It also shows the CC of cepharanthine on Huh-7 cells 50, which was 24.61 μM. This experiment was repeated three times, and the results of the three experiments were consistent or approximate.

[0065] Figure 2 The SI value (i.e., selectivity index) shown in is used to measure the ratio between cytotoxicity and antiviral activity, which is calculated by dividing CC 50 by EC 50 . The higher the SI ratio, theoretically, the more effective and safe the drug is. After calculation, the SI value of cepharanthine is 15.92, indicating its high effectiveness and safety, and it is expected to become a potential candidate drug for the treatment of SADS-CoV infectious diseases.

[0066] Example 4: Time-of-addition experiment. By qPCR and plaque assay, the inhibitory effect of cepharanthine on SADS-CoV virus infection at different drug addition stages was detected

[0067] To further explore the inhibitory effect of cepharanthine on SADS-CoV infection, the inventors conducted a time-of-addition experiment (Time-of-addition, that is, adding drugs at different stages of virus infection), observed cell morphology by electron microscopy, detected virus titer by plaque assay, and detected viral RNA by qPCR.

[0068] Experimental Method for Drug Administration Time : Seed Huh-7 cells in 24- or 48-well plates and perform the experiment 18-24 hours later. According to different stages of virus-infected cells, the time of adding drugs is divided into three groups: Entry, Post-entry, and Full-time, that is, adding drugs at the stage of virus entering cells, the stage after entry, and the whole process of virus infecting cells, respectively. The specific experimental methods are as follows: Before infecting the virus, add the drug to the Entry and Full-time groups and incubate with the cells for 2 h, then add the virus for infection (MOI = 0.1). At the time of infection, the Entry and Full-time groups are incubated with the virus, cells, and the corresponding concentration of the drug, while the Post-entry and virus-added control groups are only incubated with the virus and cells. After 2 hours of virus infection, remove the supernatant and wash once with PBS. Add drug-free medium to the Entry and virus-added control groups, while add medium containing the corresponding concentration of the drug to the Post-entry and Full-time groups. Incubate the cells at 37 °C and 5% CO2 for 48 hours, observe cell morphology by electron microscopy. Thereafter, collect cell culture supernatants and cells, and perform plaque assay and qPCR detection respectively. For visualization, the inventors provided a schematic diagram of the experimental procedure for this time-of-addition experiment, as shown in Figure 3 .

[0069] Plaque Assay Method:One day before the experiment, Huh-7 cells were seeded in a 6-well plate for 18 - 24 h. When the cell confluence reached about 80%, the experiment began. First, the collected cell culture supernatant was serially diluted 10-fold (10 -1 ~10 -6 ) with the culture medium, and the volume of each dilution was 1 mL. 1 mL of the supernatant dilutions with different dilutions were added to the 6-well plate and co-incubated with the cells at 37°C and 5% CO2 for 2 h. Then, the supernatant was removed and the cells were washed once with PBS. 2 mL of the medium containing 1% low melting point agar was added to each well. After the agar solidified, the plate was placed in an incubator at 37°C and 5% CO2 and continued to be cultured for 48 - 72 h. 1 mL of 4% tissue cell fixative (Solarbio, product number P1110) was added to each well for cell fixation for 2 h. Then, the agar and fixative in the plate were removed. 1 mL of crystal violet (Solarbio, product number) was added to each well for staining for 5 minutes. The crystal violet was removed, and the plate was rinsed with water. Then, the plaques were counted.

[0070] The virus titer calculation formula is as follows:

[0071]

[0072] Experimental Results:

[0073] The qPCR detection results of each group are as Figure 4 shown, the electron microscopy results are as Figure 5 shown, and the plaque assay results are as Figures 6 - 9 shown. Moreover, the virus titer values are shown in Table 2.

[0074] Table 2. Results of virus titer determination in the plaque assay using the supernatant of the drug treatment groups before virus entry (Entry), after virus entry (Post entry), and throughout the infection (Full time) in the drug addition time experiment

[0075]

[0076] From Figure 4 the qPCR detection results, it can be seen that adding 6.25 μM of cepharanthine throughout the process of virus infection can reduce the expression level of viral RNA in cells and can well inhibit the replication of SADS-CoV on Huh-7 cells; adding the drug before virus entry can also reduce the expression level of intracellular viral RNA and significantly inhibit virus replication.

[0077] From Figure 5It can be seen from the electron microscopy test results that compared with the virus-added control group, the degree of cytopathic effect in the Entry and Full time groups treated with the drug before virus entry and throughout the infection process was significantly reduced.

[0078] From Figures 6 - 9 the plaque assay results of -4 it can be seen that at this dilution of 10, the number of plaques in the virus-added control group was significantly more than that in the pre-entry treatment group (Entry) and the whole-process treatment (Full time) group. This indicates that the virus titer in the supernatant of the virus-added control group was significantly higher than that in the other two groups.

[0079] It can be seen from the virus titer results in Table 2 that compared with the virus-added control, the virus titer in the supernatant of the pre-entry treatment group (Entry) and the whole-infection treatment group (Full time) was significantly reduced, while there was no significant difference in the virus titer in the supernatant of the post-entry treatment group (Post entry) compared with the virus-added control group. The virus titer results in the whole-process treatment group (Full time) indicate that cepharanthine can indeed significantly inhibit the progeny proliferation of SADS-CoV on Huh-7 cells and has the effect of inhibiting the infection of SADS-CoV on Huh-7 cells; at the same time, the reduction in the virus titer in the supernatant of the pre-entry treatment group (Entry) compared with the virus-added control group also shows that cepharanthine may have the effect of inhibiting virus entry at the virus entry stage.

[0080] These results confirm that cepharanthine can significantly inhibit the replication of SADS-CoV on Huh-7 and can achieve a significant inhibitory effect at the virus entry stage.

[0081] Example 5: Detection of the inhibitory effect of cepharanthine on SADS-CoV virus infection through virus adsorption experiments and drug incubation experiments

[0082] To further explore the inhibitory effect of cepharanthine on SADS-CoV infection, the inventors conducted virus adsorption experiments, drug (i.e., cepharanthine) incubation experiments, and measured the TCID in the supernatant after incubation 50 .

[0083] Virus Adsorption Assay Method :

[0084] Huh-7 cells were seeded in 48-well plates. After 18 - 24 hours, when the cell confluence reached 80 - 90%, an adsorption experiment was carried out. First, the cells were pre-cooled at 4°C for 2 hours, and then different concentrations of drugs were added and incubated with the cells at 4°C for 2 h; the control group was incubated with a medium containing 0.1% DMSO (i.e., without drugs); then, the cell culture supernatant was discarded, and the virus solution pre-cooled at 4°C and the drug were mixed and added thereto, so that the final concentration of the drug was the corresponding incubation concentration, i.e., 3.125 μM or 6.25 μM (the control group was a mixture of the virus solution and the medium containing DMSO), and infection was carried out at 4°C (MOI = 2). After 2 hours of infection, the supernatant was removed and the cells were washed twice with pre-cooled PBS, and the cells were collected and qPCR was performed to detect the amount of viral RNA adsorbed on the cells (the method was referred to the " Virus RNA Extraction and Quantitative Real-time PCR (Quantitative Real-time PCR, qPCR)” part of the description ) in the "General Experimental Methods" described above.

[0085] Drug Incubation Assay Method :

[0086] Huh-7 cells were seeded in 48-well plates. After 18 - 24 hours, when the cell confluence reached 80 - 90%, a drug incubation experiment was carried out.

[0087] First, 6.25 μM of the drug (i.e., cepharanthine drug, as described above, first dilute the 10 mM cepharanthine stock solution to 1 mM with PBS, and then further dilute it to 6.25 μM with the medium) was used to treat the cells or the virus in groups. After incubation, virus infection (MOI = 2) was carried out. The specific operations were as follows:

[0088] The experiment was divided into three groups, namely the virus incubation group, the cell incubation group, and the virus-cell co-incubation group.

[0089] Among them, the treatment method of the virus incubation group is as follows: incubate the SADS-CoV virus with 6.25 μM of the drug alone for 2 h, without pre-treating the cells. Then, add the virus-drug incubation solution to the cells for virus infection. The treatment method of the cell incubation group is as follows: incubate the cells with 6.25 μM of the drug alone for 2 h, without pre-treating the virus. After incubation, remove the supernatant of the cell-drug incubation and wash the cells with PBS. Then, add the virus for infection. The treatment method of the virus-cell co-incubation group is as follows: incubate the cells with 6.25 μM of the drug alone for 2 h, and at the same time incubate the SADS-CoV virus with 6.25 μM of the drug alone for 2 h. After incubation, remove the supernatant of the cell-drug incubation and wash the cells with PBS, and then add the virus-drug incubation solution to it for virus infection. The control group is only incubated with a medium containing 0.1% DMSO without adding virus and drug. After 2 h of virus infection, discard the supernatant, wash the cells with PBS, add a medium containing 2% FBS, and continue to culture until 24 h. Collect the cell supernatant and perform the determination of the 50% tissue culture infective dose (TCID 50 ) assay.

[0090] TCID 50 Measurement method :

[0091] One day before the experiment, inoculate Huh-7 cells in a 96-well plate for 18 - 24 h. When the cell confluence is about 80%, start the experiment. First, dilute the collected cell culture supernatant 10-fold serially with the medium (the dilution concentration is between 10 -1 and 10 -6 ), and the volume of the liquid for each dilution is 500 μL. Add the supernatant dilutions of different dilutions to the 96-well plate, 100 μL per well, with 5 wells for each dilution. Incubate with the cells for 2 h at 37 °C and 5% CO2, then remove the supernatant and wash once with PBS. Then, add 100 μL of the medium without virus to each well and continue to culture until 48 - 72 h. The above experiment is repeated four times. Observe the cytopathic effect under the microscope, record the wells with cytopathic effect and non-cytopathic effect respectively, and calculate the TCID 50 using the Reed-Muench method (described in the reference Burleson FG, Chambers TM, Wiedbrauk DL. Virology: a laboratory manual. London: Academic Press; 1992. pp. 244–246).

[0092] Experimental Results:

[0093] In the virus adsorption experiment, the results of detecting the content of viral RNA adsorbed on cells by qPCR method are as follows Figure 10 shown; from Figure 10 it can be seen that the addition of 6.25 μM cepharanthine can significantly reduce the amount of virus adsorbed on Huh7 cells, which indicates that: at an appropriate concentration, cepharanthine can significantly inhibit the adsorption of SADS-CoV virus.

[0094] In the drug (i.e., cepharanthine) incubation experiment, the determination results of the virus titer (i.e., TCID 50 / mL) in cells after 24 hpi for each experimental group are as follows Figure 11 shown; from Figure 11 it can be seen that pre-incubating cepharanthine with cells can effectively inhibit the proliferation of virus progeny and reduce the production of infectious particles; this suggests that cepharanthine can prevent the infection of SADS-CoV virus.

[0095] Discussion

[0096] In the above examples, the inventors used SADS-CoV virus (MOI = 0.1) to infect Huh-7 cells as a virus infection model to explore the inhibitory effect of cepharanthine on SADS-CoV infection. By detecting the inhibitory activity of cepharanthine in inhibiting SADS-CoV infection in Huh-7 cells (its EC 50 = 1.546 μM), it was confirmed that cepharanthine can indeed significantly inhibit SADS-CoV virus infection, and this inhibition has an obvious dose-dependence; in addition, the cytotoxicity of cepharanthine (its CC 50 = 24.61 μM) was also detected, and based on its EC 50 and CC 50 its SI value was calculated to be 15.92, and this SI value shows its high effectiveness and safety.

[0097] In addition, it was found by adding cepharanthine treatment at different stages of virus infection that cepharanthine can significantly inhibit the infection of SADS-CoV to Huh-7 cells, and can play an obvious inhibitory role at the stage of virus entry into cells; the inhibitory effect of cepharanthine on SADS-CoV virus infection was further confirmed by plaque assay.

[0098] Furthermore, through virus adsorption experiment and drug incubation experiment, it was found that cepharanthine can significantly inhibit the adsorption of SADS-CoV virus to cells, and when pre-incubated with cells, it can effectively inhibit the proliferation of virus progeny and reduce the production of infectious particles.

[0099] These experimental results all indicate that cepharanthine can significantly inhibit the infection of SADS-CoV virus to cells, has obvious antiviral effects, and is a potential antiviral drug for the treatment of SADS-CoV infectious diseases.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of cepharanthine or its salt in the preparation of an inhibitor against porcine acute diarrhea syndrome coronavirus.

2. Use of cepharanthine or its salt in the preparation of a medicament for preventing and / or treating porcine acute diarrhea syndrome coronavirus infectious diseases.

3. The use according to claim 2, wherein The prevention and / or treatment of porcine acute diarrhea syndrome coronavirus infectious disease is to inhibit the activity of porcine acute diarrhea syndrome coronavirus.

4. The use according to claim 3, wherein The inhibition of the activity of porcine acute diarrhea syndrome coronavirus includes: inhibiting the replication of porcine acute diarrhea syndrome coronavirus and / or inhibiting the entry of porcine acute diarrhea syndrome coronavirus into cells.

5. The use according to any one of claims 1-4, wherein The salt of cepharanthine is cepharanthine hydrochloride, cepharanthine sulfate, cepharanthine acetate or cepharanthine sulfonate.

6. The use according to any one of claims 1-4, wherein The dosage form of the drug is injection, oral liquid, powder, tablet, granule, capsule, syrup, powder for injection, aqueous injection, decoction, medicinal liquor, sustained-release or controlled-release preparation, enteric-coated preparation, aerosol or suspension.

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