Application of Liriope platyphylla Saponin C in the preparation of anti-respiratory virus drugs

Abstract

The present invention provides the use of liriope muscari saponin C in the preparation of anti-respiratory virus drugs, belonging to the field of antiviral drug preparation. The liriope muscari saponin C of the present invention shows different degrees of inhibitory effects on influenza virus (H1N1) and novel coronavirus (SARS-CoV-2), showing good antiviral application prospects. Liriope muscari saponin C effectively inhibits the replication of influenza virus and the infection of SARS-CoV-2 to host by inhibiting the nuclear translocation of influenza virus vRNP complex and the binding of SARS-CoV-2 S protein and ACE2.

Description

Technical Field

[0001] The present invention belongs to the field of preparation of antiviral drugs, and particularly relates to the application of liriope muscari baily saponin C in the preparation of anti-respiratory virus drugs. Background Art

[0002] Respiratory virus infection is an important public health problem, especially in children, the elderly, and immunocompromised populations. Millions of people's lives are endangered by viral respiratory infections every year. Currently, a variety of highly contagious and pathogenic viruses to humans have been discovered, among which human coronaviruses and influenza viruses are one of the main causes of irregular outbreaks of infectious diseases.

[0003] Influenza virus is an enveloped virus of the family Orthomyxoviridae. According to the antigenicity of its nucleocapsid and matrix proteins, it is divided into four types: A, B, C, and D. Among them, influenza A virus (IAV) is the most common pathogen causing human respiratory diseases, and different subtypes such as H7N9, H9N2, H5N1, H3N2, and H1N1 prevail every year. Due to the characteristics of rapid mutation and unpredictable antigenic changes of influenza virus, vaccine production is often lagging, and it cannot provide the most timely and effective protection for the population. Therefore, drug treatment against influenza virus remains an important strategy for influenza treatment. Currently, anti-influenza virus drugs mainly include hemagglutinin inhibitors, neuraminidase inhibitors, M2 ion channel inhibitors, and viral RNA polymerase inhibitors. Since these drugs act on a single target, long-term use easily leads to the frequent emergence of drug-resistant strains, posing a huge challenge to the effectiveness of existing drugs. Therefore, it is necessary to study as many anti-influenza virus drugs acting on different targets as possible to avoid the drug resistance problem caused by using drugs acting on a single target.

[0004] Coronaviruses are spherical or pleomorphic enveloped viruses, which are single positive-strand RNA viruses, causing most upper respiratory tract infections such as the common cold and sometimes lower respiratory tract diseases such as bronchitis or pneumonia. Coronaviruses that infect humans include HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory virus coronavirus (MERS-CoV), and the novel coronavirus (SARS-CoV-2) that has been prevalent in recent years. Among them, the characteristics of the novel coronavirus (COVID-19) include symptoms such as fever, dry cough, and fatigue. The novel coronavirus is mainly transmitted through respiratory droplets and contact. Antiviral drugs such as remdesivir (an RNA polymerase inhibitor), molnupiravir (an RdRp inhibitor), and nirmatrelvir (a SARS-CoV-2 main protease inhibitor) have been used in the clinical treatment of COVID-19. However, due to the continuous emergence of drug resistance and variant strains, it is urgent to find new safe and effective specific drugs.

[0005] For thousands of years, medicinal plants have been regarded as a reliable resource for preventing and treating various diseases. Compared with synthetic drugs, herbal medicines have relatively lower costs and better compatibility with the human body. Therefore, more than 70% of the global population still relies on herbal medicines. Many chemical substances identified in various plants have shown antiviral activities, including alkaloids, flavonoids, triterpenes, anthraquinones, and lignans. Saponin chemical substances are the main active ingredients in many Chinese herbal medicines such as ginseng, bupleurum, licorice, and platycodon grandiflorum. Studies have reported that steroidal saponins have effects such as antioxidant, antidepressant, antitumor, and anti-inflammatory, and triterpenoid saponins have various biological activities such as antiviral, anti-inflammatory, and anticancer. Therefore, there is sufficient theoretical basis and practical experience to search for inhibitors with broad-spectrum anti-respiratory virus activities among natural plant chemical substances, and it has broad development prospects. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide the application of liriope muscari saponin C in the preparation of anti-respiratory virus drugs.

[0007] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0008] The present invention provides the application of liriope muscari saponin C in the preparation of antiviral drugs.

[0009] Preferably, the virus is H1N1 influenza virus or SARS-CoV-2.

[0010] Preferably, the concentration of liriope muscari saponin C for inhibiting H1N1 influenza virus is 1.10 - 22 μM.

[0011] Preferably, the concentration of Liriope muscari saponin C for inhibiting SARS-CoV-2 is 1-22 μM.

[0012] Preferably, Liriope muscari saponin C can bind to the S protein of SARS-CoV-2.

[0013] Preferably, the dosage forms of the drug include pills, tablets, powders, capsules, granules, powders, dripping pills, drops, sprays, injections, suspensions.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides the application of Liriope muscari saponin C in the preparation of antiviral drugs. The Liriope muscari saponin C of the present invention shows different degrees of inhibitory effects on influenza virus and novel coronavirus (SARS-CoV-2). Liriope muscari saponin C effectively inhibits the replication of influenza virus and the infection of SARS-CoV-2 to host by inhibiting the nuclear translocation of influenza virus vRNP complex and the binding of SARS-CoV-2 S protein and ACE2, and has good antiviral application prospects. Description of the Drawings

[0016] Figure 1 shows the inhibitory rate of Liriope muscari saponin C on influenza virus-infected MDCK cells;

[0017] Figure 2 shows the inhibitory rate of Liriope muscari saponin C on influenza virus-infected A549 cells;

[0018] Figure 3 shows the cytotoxicity of Liriope muscari saponin C to MDCK;

[0019] Figure 4 shows the cytotoxicity of Liriope muscari saponin C to A549 cells;

[0020] Figure 5 shows the study on the stage of the inhibitory effect of Liriope muscari saponin C on influenza virus, where A is the schematic diagram of the addition time of Liriope muscari saponin C; B is the virus infection inhibition rate of adding Liriope muscari saponin C at different times;

[0021] Figure 6 shows the effect of Liriope muscari saponin C on the nuclear-cytoplasmic shuttle transport of influenza virus vRNP; where A is the administration of DMSO after infecting MDCK cells with influenza virus; B is the administration of Liriope muscari saponin C (10 μmol / L) after infecting MDCK cells with influenza virus. Scale bar = 20 μm;

[0022] Figure 7is the inhibition rate of Liriope platyphylla saponin C on SARS-CoV-2 pseudovirus infecting HEK293T / hACE2 cells;

[0023] Figure 8 is the inhibition rate of Liriope platyphylla saponin C on SARS-CoV-2 pseudovirus infecting Vero E6 / hACE2 cells;

[0024] Figure 9 is the cytotoxicity of Liriope platyphylla saponin C on HEK293T / hACE2;

[0025] Figure 10 is the cytotoxicity of Liriope platyphylla saponin C on Vero E6 / hACE2;

[0026] Figure 11 is the inhibitory effect of Liriope platyphylla saponin C on SARS-CoV-2 pseudovirus infection;

[0027] Figure 12 is the inhibitory effect diagram of Liriope platyphylla saponin C on SARS-CoV-2 pseudovirus infection after being added at different infection stages;

[0028] Figure 13 is the interaction diagram of Liriope platyphylla saponin C inhibiting the interaction between SARS-CoV-2 S protein and hACE2 receptor;

[0029] Figure 14 is the dose-response curve diagram of the effect of Liriope platyphylla saponin C on the biological activity of receptor hACE2;

[0030] Figure 15 is Liriope platyphylla saponin C inhibiting cell fusion mediated by SARS-CoV-2 S protein (magnification = 10×10 times);

[0031] Figure 16 is the relative fusion rate diagram under the action of Liriope platyphylla saponin C. Detailed implementation mode

[0032] The present invention provides the application of Liriope platyphylla saponin C in the preparation of antiviral drugs.

[0033] In the present invention, the virus is influenza virus or SARS-CoV-2.

[0034] In the present invention, the preferred concentration of Liriope platyphylla saponin C for inhibiting H1N1 influenza virus is 1.10 - 22 μM, more preferably 2.38 - 21 μM, and even more preferably 20 μM.

[0035] Preferably, the concentration of Liriope muscari saponin C for inhibiting SARS-CoV-2 is preferably 1-22 μM, more preferably 7.18-21 μM, and even more preferably 20 μM.

[0036] Preferably, Liriope muscari saponin C can bind to the S protein of SARS-CoV-2 and prevent the virus from spreading between target cells.

[0037] Preferably, the dosage forms of the drug include pills, tablets, powders, capsules, granules, powders, dripping pills, drops, sprays, injections, suspensions.

[0038] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Experimental Materials and Instruments

[0040] 1) Cells, Viruses and Plasmids

[0041] HEK-293T (human embryonic kidney cells), HEK293T cell line stably expressing human ACE2 (hACE2) receptor and Vero E6 cell line are from Shanghai Institute of Immunology and Infection, Chinese Academy of Sciences;

[0042] MDCK cell line is from Lanzhou Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences;

[0043] A549 (human non-small cell lung cancer cells) is from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences;

[0044] Luciferase reporter virus PR8-NS-Nluc is from Lanzhou Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences;

[0045] Plasmid pGIPZ-empty expressing GFP is purchased from OpenBiosystems;

[0046] Plasmid expressing the envelope protein of novel coronavirus: SARS-CoV-2-S (pVAX1-nCoVS) plasmid is from Shanghai Institute of Immunology and Infection, Chinese Academy of Sciences;

[0047] SARS-CoV-2-S pseudovirus particles and VSV-G pseudovirus particles are from School of Public Health, Lanzhou University.

[0048] 2) Compounds

[0049] Liriope muscari saponin C (HY-N5055) is purchased from MCE Company (MedChemExpress, USA), with a purity of over 99%, dissolved in dimethyl sulfoxide (DMSO) solvent and stored at a concentration of 10 mM.

[0050] 3) Experimental reagents

[0051] Table 1 Cell experimental reagents

[0052]

[0053] Table 2 Main experimental reagents

[0054]

[0055]

[0056] Table 3 Main experimental instruments

[0057]

[0058] Example 1: Total administration of Liriope platyphylla saponin C to study its inhibitory effect on influenza virus-infected MDCK cells and A549 cells

[0059] Using the luciferase reporter virus PR8-NS-Nluc, the replication of influenza virus was quantitatively evaluated by detecting the expression level of the tag protein luciferase gene NanoLuc Luciferase (NLuc) in infected cells, so as to explore the anti-influenza virus effect of Liriope platyphylla saponin C. The specific method is as follows:

[0060] (1) MDCK cells in the logarithmic growth phase growing in DMEM medium containing 10% fetal bovine serum were evenly inoculated into 96-well plates at a density of 2×10 4 cells / well, and cultured overnight in an incubator at 37°C with 5% CO2 until the cells grew to about 80%, and then the infection experiment was carried out.

[0061] (2) The virus solution (MOI = 0.01) was prepared with serum-free DMEM medium and added to the 96-well plates at 50 μL / well. The plates were placed in a cell incubator at 37°C with 5% CO2 and incubated for 1 h.

[0062] (3) The supernatant was aspirated, and Liriope platyphylla saponin C was diluted into 5 gradients with final concentrations of 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM with serum-free DMEM medium containing TPCK-trypsin (1 μg / mL). Then the corresponding diluted solutions of Liriope platyphylla saponin C were added to the wells, and a virus control group without compound treatment was set. The incubation was continued in a cell incubator at 37°C with 5% CO2 for 24 h.

[0063] (4) Subsequently, luciferase was added for lysis and luminescence, and a multifunctional microplate reader was used to detect the chemiluminescence value. Compared with the control group, the inhibition rate was calculated to determine its IC50 value. Inhibition rate (%) = (1 - chemiluminescence value of the drug-treated group / chemiluminescence value of the control group) × 100%, and GraphPad Prism 8.0 was used for plotting and calculating the IC50.

[0064] Experimental results: As Figure 1 shown. It can be Figure 1 seen that on MDCK cells, the inhibition rate of liriope muscari saponin C against influenza virus infection increased with the increase of concentration, and its IC50 = 1.10 μM.

[0065] In addition, the anti-viral infection experiment of liriope muscari saponin C was also carried out in A549 cells. 2×10 4 cells were seeded in each well of a 96-well plate and cultured until the cells grew to about 80%. Other operations were basically the same as those in the above-mentioned MDCK cell infection experiment.

[0066] Experimental results: As Figure 2 shown. It can be Figure 2 seen that on A549 cells, the inhibition rate of liriope muscari saponin C against influenza virus increased with the increase of concentration, and the IC50 was 2.38 μM.

[0067] Example 2 Detection of the toxicity of liriope muscari saponin C to MDCK cells and A549 cells by CCK8 method

[0068] MDCK cells in the logarithmic growth phase growing in DMEM culture medium containing 10% fetal bovine serum were evenly seeded in a 96-well plate at a density of 2×10 4 cells / well and cultured overnight in an incubator at 37°C with 5% CO2 until the cells grew to about 80%. Liriope muscari saponin C was diluted with DMEM diluent into 8 concentration gradients with a starting concentration of 100 μM by 2-fold serial dilution. The MDCK cells were incubated with different concentrations of liriope muscari saponin C at 37°C for 48 h, and then the cell viability was detected using CCK8 (completely consistent with the drug administration process). The specific operation was as follows. Under light-proof conditions, 10 μL of CCK8 reagent was added to each well and incubated at 37°C in the dark for 1 h. The wells containing only DMEM culture medium with 10% CCK8 reagent were used as blank controls. The absorbance value of the cells at a wavelength of 450 nm was detected by a microplate reader. Calculation of cell viability: Cell viability (%) = [OD (drug-treated) - OD (blank)] / [OD (control) - OD (blank)] × 100%.

[0069] Experimental results: As Figure 3 shown. It can be Figure 3It can be seen that the half-toxic concentration (CC50) of Liriope muscari saponin C on MDCK cells = 54.92 μM.

[0070] In addition, cytotoxicity detection was also carried out in A549 cells. 2×10 4 cells were seeded in each well of a 96-well plate and cultured until the cells grew to about 80%. Liriope muscari saponin C was diluted with DMEM diluent into 9 concentration gradients with a starting concentration of 100 μM and a 2-fold serial dilution. Other operations were basically the same as those for the above-mentioned MDCK cell cytotoxicity detection.

[0071] Experimental results: As Figure 4 shown. It can be Figure 4 seen that the half-toxic concentration (CC50) of Liriope muscari saponin C on A549 cells = 25.04 μM.

[0072] The above results indicate that the effective inhibitory concentration of Liriope muscari saponin C against influenza virus infection is lower than its cytotoxic concentration, showing good cell safety.

[0073] Example 3 Determination of the addition time of Liriope muscari saponin C

[0074] To clarify the effect of Liriope muscari saponin C administered at different time points during the influenza replication cycle on influenza virus proliferation, a Time-of-addition assay was performed. The specific method is as follows: Logarithmic-phase MDCK cells growing in DMEM culture medium containing 10% fetal bovine serum were evenly seeded in a 96-well plate at a density of 2×10 4 cells / well and cultured overnight in an incubator at 37°C with 5% CO2 until the cells grew to about 80%, and then an infection experiment was carried out. The cells were infected with a virus solution (MOI = 0.05) prepared with serum-free DMEM medium, and the plate was incubated in a cell incubator at 37°C with 5% CO2 for 2 h. The time point when the plate was transferred to the incubator was set as 0 h.p.i. At the designated time intervals (0 - 2, 2 - 4, 4 - 6, 6 - 8, 8 - 10 hours and whole-course administration, where the whole-course administration was from 2 hours before virus infection of MDCK cells to 24 hours after infection), the cells were treated with DMSO or 10 μM Liriope muscari saponin C, and then the cells were incubated with fresh medium until 24 h after infection. The luciferase activity in the cell lysate was detected, and the inhibition rate was calculated based on the chemiluminescence value of the control group to determine the stage at which Liriope muscari saponin C inhibits influenza virus.

[0075] Experimental results: As Figure 5 shown, adding Liriope muscari saponin C at 2 - 4 h and 4 - 6 h showed a relatively high inhibition rate, which was consistent with the inhibitory effect of whole-course administration, indicating that Liriope muscari saponin C plays a role in the early stage of replication after the virus enters the cells.

[0076] Example 4: Effect of Liriope platyphylla Saponin C on the Nuclear-cytoplasmic Shuttle Transport of Influenza Virus vRNP

[0077] The shuttle transport of the influenza virus vRNP complex between the nucleus and the cytoplasm plays an important role in virus replication. In this study, at different time points after influenza virus infection, the indirect immunofluorescence method was used to label the NP protein to localize the intracellular position of vRNP, and then by comparing the treatment with Liriope platyphylla Saponin C or DMSO, whether the compound has an effect on the nuclear-cytoplasmic shuttle transport of influenza virus vRNP was observed. The specific methods are as follows:

[0078] (1) Prepare glass slides for cell culture: Take out ordinary clean coverslips, soak them in 70% ethanol for 5 min, then dry them in a sterile laminar flow hood or wash them three times with cell culture grade PBS / 0.9% NaCl solution, and finally wash them once with cell culture medium;

[0079] (2) Seed cells on glass slides: Place the prepared glass slides in a 12-well plate, and inoculate the MDCK cells in the logarithmic growth phase grown in DMEM medium containing 10% fetal bovine serum into the 12-well plate at a density of 3×10 5 cells / well, and culture them overnight in an incubator at 37°C with 5% CO2;

[0080] (3) Treatment: When the cells grow to about 80%, perform the infection experiment. Add the virus solution prepared with serum-free DMEM medium (MOI = 5), discard the supernatant after infecting at 37°C for 1 hour, and add Liriope platyphylla Saponin C (10 μM) or DMSO diluted with serum-free DMEM medium containing TPCK-trypsin (1 μg / mL). Discard the supernatant at 2 h, 4 h, 6 h, and 8 h respectively. Take out the glass slides at different time points and wash them 3 times with PBS (5 min / time, either on a shaker or static, the same below);

[0081] (4) Fixation: Add 4% paraformaldehyde to fix the cells at room temperature. After fixing for 10 min, add PBS to wash 3 times. Fix the glass slides at different time points and place them at 4°C. After the 8 h glass slides are fixed, proceed to the following steps;

[0082] (5) Blocking: Add QuickBlock TM immunostaining blocking solution containing an appropriate amount of Triton-X 100, block at room temperature for 30 min, and remove the blocking solution for antibody incubation;

[0083] (6) Primary antibody incubation: Add the primary antibody diluted with QuickBlock TMThe Influenza A virus NP antibody (nanobody with human Fc tag, 1:200) diluted with the primary antibody dilution solution for immunostaining was incubated overnight at 4°C. The slides were washed 3 times with PBS;

[0084] (7) Secondary antibody incubation: Add FITC-labeled goat anti-human IgG (H+L) diluted with the secondary antibody dilution solution for immunofluorescence staining, and incubate in the dark at room temperature for 1 h. The slides were washed 3 times with PBS; TM The Influenza A virus NP antibody (nanobody with human Fc tag, 1:200) diluted with the primary antibody dilution solution for immunostaining was incubated overnight at 4°C. The slides were washed 3 times with PBS;

[0085] (8) Mounting: Drop the anti-fluorescence quenching mounting solution containing DAPI on the glass slide for mounting. Take out the cell slides from the well plate and invert them on the mounting agent for DAPI staining and mounting;

[0086] (9) Observe and take pictures using a confocal microscope.

[0087] Experimental results: As Figure 6 shown, as can be seen from A in Figure 6 , in the DMSO group, the vRNP complex was mainly concentrated in the cytoplasm 2 h after infection. At 4 h after infection, vRNP began to transfer to the nucleus. By 8 h after infection, vRNP was distributed in both the cytoplasm and the nucleus. As Figure 6 shown in B in

[0088] compared with the DMSO group, after treatment with liriope polysaccharide C, the vRNP complex was mostly concentrated in the cytoplasm, and the difference was most obvious at 8 h after infection, indicating that liriope polysaccharide C can inhibit the nuclear-cytoplasmic shuttle transport of vRNP after virus infection.

[0089] In summary, liriope polysaccharide C can effectively inhibit influenza virus infection, with an effective concentration of 1.25 - 20 μM and relatively low cytotoxicity. It has the potential to be an effective anti-influenza virus infection drug and has broad application prospects.

[0090] HEK293T / hACE2 cells in the logarithmic growth phase grown in DMEM medium containing 10% fetal bovine serum were seeded at a density of 2×10 4Cells were evenly inoculated into a 96-well plate at a density of [number of cells / holes], and cultured overnight in an incubator at 37°C with 5% CO2 until the cells grew to about 80%. Then, an infection experiment was conducted. Liriope spicata saponin C was diluted with DMEM diluent to four concentration gradients of 20 μM, 10 μM, 5 μM, and 1 μM. Different concentrations of Liriope spicata saponin C were mixed evenly with the SARS-CoV-2 S protein pseudovirus solution. The control group was added with a mixture of an equal volume of DMEM diluent and the pseudovirus solution. Incubate at 37°C for 48 h, then discard the medium, and then add luciferase to lyse and emit light. The chemiluminescence value was detected using a multifunctional microplate reader. Compared with the control group, the inhibition rate was calculated to determine its IC50 value. Inhibition rate (%) = (1 - chemiluminescence value of the drug-treated group / chemiluminescence value of the control group) × 100%. GraphPad Prism 8.0 was used to plot the graph and calculate the half-maximal inhibitory concentration (IC50).

[0091] Experimental results: As Figure 7 shown. From Figure 7 it can be seen that on HEK293T / hACE2 cells, the inhibition rate of Liriope spicata saponin C on the infection of SARS-CoV-2 pseudovirus increased with the increase of concentration, and its half-maximal inhibitory concentration (IC50) = 1.50 μM.

[0092] In addition, an infection experiment was also conducted in Vero E6 / hACE2 cells. 1.5×10 4 cells were inoculated into each well of a 96-well plate and cultured until the cells grew to about 80%. Other operations were basically the same as those in the above HEK293T / hACE2 cell infection experiment.

[0093] Experimental results: As Figure 8 shown. From Figure 8 it can be seen that on Vero E6 / hACE2 cells, the inhibition rate of Liriope spicata saponin C on the SARS-CoV-2 pseudovirus increased with the increase of concentration, and its half-maximal inhibitory concentration (IC50) = 7.18 μM.

[0094] Example 6 Detection of the toxicity of Liriope spicata saponin C to HEK293T / hACE2 cells and Vero E6 / hACE2 by CCK8 method

[0095] HEK293T / hACE2 cells in the logarithmic growth phase growing in DMEM culture medium containing 10% fetal bovine serum were seeded at 2×10 4Cells were evenly inoculated into a 96-well plate at a density of [number] / well and cultured overnight at 37°C in an incubator containing 5% CO2. When the cell confluence reached about 80%, Liriope spicata saponin C was diluted with DMEM diluent into 8 concentration gradients with the initial concentration of 100 μM and a 2-fold serial dilution. HEK293T / hACE2 cells were incubated with different concentrations of Liriope spicata saponin C at 37°C for 48 h, and then the cell viability was detected using CCK8 (completely consistent with the drug administration process). The specific operation was as follows: Under light-proof conditions, 10 μL of CCK8 reagent was added to each well and incubated at 37°C in the dark for 2 h. The well containing only DMEM culture medium with 10% CCK8 reagent was used as a blank control. The absorbance of the cells at a wavelength of 450 nm was detected by an enzyme-linked immunosorbent assay (ELISA) reader. Cell viability calculation: Cell viability (%) = [OD (drug added) - OD (blank)] / [OD (control) - OD (blank)] × 100%.

[0096] Experimental results: As Figure 9 shown. From Figure 9 it can be seen that the half-maximal cytotoxic concentration (CC50) of Liriope spicata saponin C on HEK293T / hACE2 cells = 69.01 μM.

[0097] In addition, cytotoxicity detection was also carried out in Vero E6 / hACE2 cells. 1.5×10 4 cells were inoculated into each well of a 96-well plate and cultured until the cell growth reached about 80%. Liriope spicata saponin C was diluted with DMEM diluent into 8 concentration gradients with the initial concentration of 200 μM and a 2-fold serial dilution. Other operations were basically the same as those for the cytotoxicity detection of HEK293T / hACE2 cells above.

[0098] Experimental results: As Figure 10 shown. From Figure 10 it can be seen that the half-maximal cytotoxic concentration (CC50) of Liriope spicata saponin C on Vero E6 / hACE2 cells = 106.4 μM.

[0099] The above results indicate that the effective inhibitory concentration of Liriope spicata saponin C against SARS-CoV-2 pseudovirus infection is much lower than its cytotoxic concentration, showing good cell safety.

[0100] Example 7 Liriope spicata saponin C inhibits SARS-CoV-2 pseudovirus entry specifically

[0101] To identify whether Liriope spicata saponin C can inhibit SARS-CoV-2 infection, the inhibitory effect of Liriope spicata saponin C on SARS-CoV-2 pseudovirus was detected. The specific method was as follows:

[0102] (1) Logarithmic growth phase HEK293T / hACE2 cells grown in DMEM culture medium containing 10% fetal bovine serum were evenly inoculated into 96-well plates at a density of 2×10 4 cells / well, placed in an incubator at 37°C with 5% CO2 overnight, and the experiment was carried out when the cells grew to about 80%.

[0103] (2) Liriope muscari saponin C was diluted into 4 gradients with final concentrations of 20 μM, 10 μM, 5 μM, and 1 μM with DMEM diluent, and then the corresponding concentrations of Liriope muscari saponin C were mixed with SARS-CoV-2 pseudovirus solution to co-infect target cells. The control wells were added with a mixture of an equal volume of DMEM diluent and pseudovirus solution.

[0104] (3) VSV-G pseudovirus was used as a control for the infection experiment to verify the specificity of Liriope muscari saponin C against SARS-CoV-2. The experimental procedure for VSV-G pseudovirus as a control was the same as above.

[0105] (4) After incubation in an incubator at 37°C with 5% CO2 for 48 h, it was taken out, and an equal volume of luciferase lysis luminescence was added to the wells in the dark. The chemiluminescence value was detected using a multifunctional microplate reader, and the relative infection rate (%) was calculated according to the control group: (chemiluminescence value of the drug-added group / chemiluminescence value of the control group)×100%.

[0106] Results: As Figure 11 shown, Liriope muscari saponin C had a weak inhibitory effect on VSV-G pseudovirus, far lower than its activity against SARS-CoV-2. Liriope muscari saponin C did not have antiviral activity against VSV-G pseudovirus at the maximum concentration of 20 μM, and even the relative infection rate increased slightly. This result indicates that Liriope muscari saponin C has specificity against SARS-CoV-2 infection.

[0107] Example 8 Liriope muscari saponin C inhibits SARS-CoV-2 pseudovirus invasion at an early stage

[0108] To clarify the stage at which Liriope muscari saponin C exerts its anti-SARS-CoV-2 infection effect, 10 μM of the compound was added to 293T-hACE2 cells at -1 h (before pseudovirus infection), 0 h (at the time of infection), and 2 h (after infection) respectively, and the luciferase activity in the cell lysate was detected after 48 h, and the inhibition rate was calculated according to the chemiluminescence value of the control group.

[0109] Results: As Figure 12As shown, when Liriope platyphylla saponin C was added before infection, that is, the drug was incubated with cells for 1 h and then SARS-CoV-2 pseudovirus was added, it showed the highest inhibition rate, which was better than the inhibition rate when the drug was added during infection. The inhibition rate when the drug was added 2 h after pseudovirus infection was lower than that when the drug was added before and during infection. This result indicates that the inhibition of SARS-CoV-2 pseudovirus invasion of target cells by the compound mainly occurs in the early stage of infection.

[0110] Example 9 Detection of the effect of Liriope platyphylla saponin C on the binding of S protein and hACE2 by ELISA

[0111] Using CoviDrop TM Experiment was carried out using the SARS-CoV-2 Spike-ACE2 Binding Inhibitor Screening Rapid Kit (D-1004). The specific steps are as follows:

[0112] (1) Buffer preparation: Add 26 mL of 10× washing solution to 234 mL of distilled water to prepare 1× washing solution; Dilute the binding detection solution and 1× washing solution to the working concentration according to the ratio of 1:2000; Dilute the ACE2 protein and detection buffer at a ratio of 1:1000 to prepare ACE2 working solution; Dilute the positive control with the detection buffer into a strong positive control solution (1 mg / mL) and a weak positive control solution (100 μg / mL);

[0113] (2) Sampling of Liriope platyphylla saponin C: Set up control groups and add samples according to Table 4;

[0114] Table 4 Sampling table of the SARS-CoV-2 Spike-ACE2 Binding Inhibitor Screening Kit

[0115]

[0116] (3) Incubation: Cover the microplate tightly with tin foil and incubate at room temperature for 15 min;

[0117] (4) Sampling of ACE2 working solution: After incubating for 15 min, add 50 μL of detection buffer to the blank well, and add 50 μL of ACE2 working solution to each of the remaining wells;

[0118] (5) Incubation: Cover the microplate tightly with tin foil again and incubate at 37 °C for 45 min;

[0119] (6) Plate washing: Discard the solution in the wells, add 150 μL of 1× washing solution to each well using a multi-channel pipette, shake the plate for 45 s, and repeat 3 times;

[0120] (7) Sampling of binding detection solution: Add 50 μL of binding detection solution to each well using a multi-channel pipette;

[0121] (8) Incubation: Cover the microplate tightly with aluminum foil and incubate at room temperature for 20 min;

[0122] (9) Plate washing: Discard the solution in the wells, add 150 μL of 1× washing buffer to each well using a multi-channel pipette, shake the plate for 45 s, and repeat 5 times;

[0123] (10) Addition of developing solution: Add 100 μL of developing solution to each well using a multi-channel pipette;

[0124] (11) Incubation: Incubate away from direct light at room temperature until the solution turns blue;

[0125] (12) Addition of stop solution: Add 100 μL of stop solution to each well, gently shake to mix the solution, and immediately measure the OD value after the reaction is complete;

[0126] (13) Measurement of OD value: Measure the OD value using a multi-functional microplate reader (Tecan infinite 200, TECAN). The measurement wavelength is 450 nm and the reference wavelength is 655 nm;

[0127] (14) Calculation of inhibition rate: Inhibition rate = [1 - (OD of compound sample - OD of blank) / (OD of negative control - OD of blank)] × 100%.

[0128] Experimental results: As Figure 13 shown, compared with the control group, Liriope platyphylla saponin C can block the binding of S protein to the receptor hACE2. The inhibitory effect is positively correlated with the concentration.

[0129] Example 10 Liriope platyphylla saponin C has no effect on the biological function of hACE2 receptor

[0130] To explore the potential effect of Liriope platyphylla saponin C on the hACE2 receptor, a R & D Systems angiotensin-converting enzyme 2 (ACE2) inhibitor screening kit (P0320S) was used to determine the inhibitory effect of the compound on the protease activity of hACE2. The specific steps are as follows:

[0131] (1) Sample preparation: Prepare concentration gradients of Liriope platyphylla saponin C with final concentrations of 500 μM, 100 μM, 20 μM, 4 μM, 0.8 μM, and 0.16 μM using Assay Buffer;

[0132] (2) Positive control preparation: Dilute the positive control inhibitor MLN-4760 to a final concentration of 0.1 μM using Assay Buffer;

[0133] (3) Preparation of Assay Reagent: According to the sample volume, mix Assay Buffer and ACE2 Enzyme in a centrifuge tube at a ratio of 92:1 and mix well;

[0134] (4) Sample addition: Add the detection reagents and samples to the 96-well black plate in sequence according to Table 5 below and mix well. Then, use a multi-channel pipette to add 2 μL of Substrate to each well and mix quickly;

[0135] Table 5 Sample addition table for ACE2 inhibitor screening kit

[0136]

[0137] (5) Incubation: Transfer the 96-well plate to an incubator at 37 °C and incubate in the dark for 45 min;

[0138] (6) Measurement of fluorescence value: Set the multi-functional microplate reader program with an excitation wavelength of 325 nm and an emission wavelength of 393 nm;

[0139] (7) Calculate the relative inhibition rate: Calculate the average values of each sample well, 100% enzyme activity control well, positive control well, and blank control well, and record them as RFU 样品 , RFU 100％酶活性对照 , RFU 阳性对照 , RFU 空白对照 . Relative inhibition rate (%) = (RFU 100％酶活性对照 - RFU 样品 ) / (RFU 100％酶活性对照 - RFU 空白对照 ) × 100%;

[0140] (8) Calculate IC50: Use GraphPad Prism 8.0 to plot a graph and calculate the IC50 of liriope muscari baily saponin C.

[0141] Experimental results: As Figure 14 shown, the IC50 of liriope muscari baily saponin C is 812.4 μM. At a concentration below 20 μM, the inhibition rate of liriope muscari baily saponin C on the protease activity of hACE2 is almost 0. When the concentration increases to 100 μM, the inhibition rate of liriope muscari baily saponin C is still relatively low, not exceeding 10%. This result indicates that the compound has no obvious inhibitory effect on the hACE2 receptor at the concentration used in the cell experiment, and it can be speculated that its anti-SARS-CoV-2 activity is mainly achieved by targeting the S protein of SARS-CoV-2.

[0142] Example 11 Effect of liriope muscari baily saponin C on cell fusion mediated by the S protein of SARS-CoV-2

[0143] Co-culture effector cells (HEK293T / SARS-CoV-2S / GFP) expressing the S protein of SARS-CoV-2 and GFP and target cells (HEK293T / hACE2) expressing the hACE2 receptor on the cell surface, so that the S protein mediates cell-cell fusion by recognizing hACE2. When effector cells fuse with target cells to form syncytia, the fused cells are usually twice as large as unfused cells, and due to the diffusion of GFP from one effector cell to the target cell, the fluorescence intensity of the fused cells will weaken, thereby detecting the effect of Liriope spicata saponin C on cell fusion. The specific method is as follows:

[0144] (1) Seed HEK293T cells in a 6-well plate at a density of 4×10 4 cells / mL and culture overnight in a 37°C cell culture incubator containing 5% CO2;

[0145] (2) When the cell confluence reaches about 80%, discard the cell supernatant, and re-add 2 mL of pre-warmed DMEM complete medium to each well. In the experimental group, mix DNA (pVAX1-nCoVS and pGIPZ-empty) with lipo8000 transfection reagent at a mass-to-volume ratio of 1:2 and co-transfect into HEK293T cells. The negative control group is transfected with pGIPZ-empty only according to the same method;

[0146] (3) After 8 h of transfection, discard the supernatant and re-add fresh DMEM complete medium;

[0147] (4) After 24 h of transfection, seed HEK293T-hACE2 cells in a 96-well transparent cell culture plate and culture in a 37°C cell culture incubator containing 5% CO2;

[0148] (5) After 48 h of transfection, take out the 6-well plate, discard the supernatant, resuspend and count the effector cells (HEK293T / SARS-CoV-2S / GFP and HEK293T / GFP cells) using DMEM complete medium. Dilute Liriope spicata saponin C into 3 concentration gradients of 5 μM, 10 μM, and 20 μM with DMEM diluent, then incubate HEK293T / SARS-CoV-2S / GFP effector cells with different concentrations of Liriope spicata saponin C for 30 min, and then add 100 μL of cell-drug mixture at a concentration of 2×10 4 cells / mL to the target cells. The positive control group adds an equal volume of HEK293T / SARS-CoV-2S / GFP effector cell suspension, and the negative control group adds an equal volume of HEK293T / GFP effector cell suspension;

[0149] After co-culturing at 37°C for 6 h, the formation of syncytia was observed under an inverted fluorescence microscope, photographed, and the cell fusion rate was counted and calculated using Image pro plus software. The cell fusion rate (%) = (E - N) / (P - N) × 100%; E is the proportion of syncytia in the experimental group among the total number of cells, P is the proportion of syncytia in the positive control group among the total number of cells, and N represents the proportion of syncytia in the negative control group among the total number of cells.

[0150] Experimental results: As Figure 15 shown, as Figure 15 can be seen, liriope saponin C significantly inhibited the fusion between HEK293T / SARS-CoV-2S / GFP cells and target cells HEK293T / hACE2, resulting in a concentration-gradient decrease in syncytium formation. At the same time, no formation of fusion vesicles was observed between HEK293T / GFP cells that did not express the S protein in the control cells and target cells HEK293T / hACE2. Further calculation of the relative fusion rate showed that (the results are shown in detail in Figure 16 ), liriope saponin C decreased the cell fusion rate in a dose-dependent manner.

[0151] These results indicate that liriope saponin C can effectively inhibit SARS-CoV-2 S protein-mediated cell fusion and prevent the spread of the virus between target cells.

[0152] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of broadleaf ophiopogon saponin C in the preparation of antiviral drugs; The virus is H1N1 influenza virus; The concentration of Ophiopogon japonicus saponin C to inhibit H1N1 influenza virus is 1.10~22µM.

2. The use according to claim 1, characterized in that The dosage forms of the medicine include pills, tablets, powders, capsules, granules, powders, dropping pills, drops, sprays, injections, and suspensions.

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