Application of liriope latifolia saponin C in preparation of anti-respiratory virus drugs

Abstract

The invention provides application of liriope latifolia saponin C in preparation of anti-respiratory virus drugs, and belongs to the field of preparation of antiviral drugs. The liriope latifolia saponin C disclosed by the invention shows different degrees of inhibition effects on influenza virus (H1N1) and novel coronavirus (SARS-CoV-2), and shows a relatively good antiviral application prospect. By inhibiting nuclear translocation of an influenza virus vRNP complex and combination of SARS-CoV-2S protein and ACE2, the liriope broadleaf saponin C effectively inhibits replication of the influenza virus and infection of SARS-CoV-2 to a host.

Description

Technical Field

[0001] The invention belongs to the field of antiviral drug preparation, and in particular relates to the application of broad-leaved ophiopogon saponin C in the preparation of anti-respiratory virus drugs. Background Art

[0002] Respiratory viral infections are a major public health issue, especially in children, the elderly and people with weakened immune systems, with millions of people dying every year from viral respiratory infections. A variety of highly contagious and pathogenic viruses have been discovered, among which human coronaviruses and influenza viruses are one of the main causes of sporadic outbreaks of infectious diseases.

[0003] Influenza virus is an enveloped virus of the family Orthomyxoviridae. It is divided into four types: A, B, C, and D according to the different antigenicity of its nucleocapsid and matrix protein. Among them, influenza A virus (IAV) is the most common pathogen causing human respiratory diseases. Different subtypes such as H7N9, H9N2, H5N1, H3N2, and H1N1 are prevalent every year. Due to the rapid mutation of influenza virus and the unpredictable antigenic changes, vaccine production often lags behind and cannot provide the most timely and effective protection for the population. Therefore, anti-influenza virus drug treatment is still an important strategy for influenza treatment. At present, 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 can easily lead to the frequent emergence of drug-resistant strains, which poses a huge challenge to the effectiveness of existing drugs. Therefore, it is necessary to study as many anti-influenza virus drugs that act on different targets as possible to avoid the drug resistance problem caused by the use of drugs that act on a single target.

[0004] Coronaviruses are spherical or pleomorphic enveloped viruses, single positive-strand RNA viruses that cause 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 new coronavirus (SARS-CoV-2) that has been prevalent in recent years. Among them, the new coronavirus (COVID-19) is characterized by symptoms such as fever, dry cough, and fatigue. The new coronavirus is mainly transmitted through respiratory droplets and contact. Antiviral drugs such as remdesivir (RNA polymerase inhibitor), monoclavir (RdRp inhibitor), and namatevir (SARS-CoV-2 main protease inhibitor) have been used in the clinical treatment of COVID-19, but due to the continuous emergence of drug resistance and variants, it is urgent to find new safe and effective specific drugs.

[0005] For thousands of years, medicinal plants have been considered a reliable resource for the prevention and treatment of a variety of diseases. Compared with synthetic drugs, herbal medicines are relatively low in cost and more compatible with the human body, so more than 70% of the world's population still relies on herbal medicines. Many chemicals identified in various plants have shown antiviral activity, including alkaloids, flavonoids, triterpenes, anthraquinones and lignans. Saponin chemicals are the main active ingredients in many Chinese herbal medicines such as ginseng, bupleurum, licorice, and platycodon. Studies have reported that steroidal saponins have antioxidant, antidepressant, antitumor and anti-inflammatory effects, and triterpenoid saponins have multiple biological activities such as antiviral, anti-inflammatory, and anticancer. Therefore, the search for inhibitors with broad-spectrum anti-respiratory virus activity in natural plant chemicals has sufficient theoretical basis and practical experience, and has broad development prospects. Summary of the invention

[0006] In view of this, the object of the present invention is to provide the use of broad-leaved ophiopogon saponin C in the preparation of anti-respiratory virus drugs.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides application of broad-leaved ophiopogon saponin C in preparing antiviral drugs.

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

[0010] Preferably, the concentration of Ophiopogon japonicus saponin C used to inhibit H1N1 influenza virus is 1.10-22 μM.

[0011] Preferably, the concentration of Ophiopogon japonicus saponin C used to inhibit SARS-CoV-2 is 1 to 22 μM.

[0012] Preferably, Ophiopogon japonicus saponin C is able to bind to the S protein of SARS-CoV-2.

[0013] Preferably, the dosage form of the drug includes pills, tablets, powders, capsules, granules, powders, pellets, drops, sprays, injections, and suspensions.

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

[0015] The present invention provides an application of broadleaf ophiopogonin C in the preparation of antiviral drugs. The broadleaf ophiopogonin C of the present invention exhibits different degrees of inhibitory effects on influenza virus and novel coronavirus (SARS-CoV-2). The broadleaf ophiopogonin C effectively inhibits the replication of influenza virus and the infection of SARS-CoV-2 to the host by inhibiting the nuclear translocation of the influenza virus vRNP complex and the binding of SARS-CoV-2S protein and ACE2, and has good antiviral application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the inhibition rate of Ophiopogon japonicus saponin C on influenza virus infection of MDCK cells;

[0017] Figure 2 is the inhibition rate of Ophiopogon japonicus saponin C on influenza virus infection of A549 cells;

[0018] Figure 3 The cytotoxicity of Ophiopogon japonicus saponin C to MDCK cells;

[0019] Figure 4 The cytotoxicity of Ophiopogon japonicus saponin C to A549 cells;

[0020] Figure 5 This is a study on the stages of the inhibitory effect of broad-leaved ophiopogonin C on influenza virus, where A is a schematic diagram of the time of adding broad-leaved ophiopogonin C; B is the virus infection inhibition rate of broad-leaved ophiopogonin C at different times of addition;

[0021] Figure 6 The effect of Ophiopogon japonicus saponin C on the nuclear-cytoplasmic shuttling of influenza virus vRNP; A is MDCK cells infected with influenza virus and then given DMSO; B is MDCK cells infected with influenza virus and then given Ophiopogon japonicus saponin C (10 μmol / L). Scale bar = 20 μm;

[0022] Figure 7is the inhibition rate of Ophiopogon japonicus saponin C on SARS-CoV-2 pseudovirus-infected HEK293T / hACE2 cells;

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

[0024] Fig. 9 The cytotoxicity of Ophiopogon japonicus saponin C to HEK293T / hACE2;

[0025] Fig.10 The cytotoxicity of Ophiopogon japonicus saponin C to Vero E6 / hACE2;

[0026] Fig.11 The inhibitory effect of Ophiopogon japonicus saponin C on SARS-CoV-2 pseudovirus infection;

[0027] Fig.12 This is a graph showing the inhibitory effect of Ophiopogon japonicus saponin C on SARS-CoV-2 pseudovirus infection after adding it at different stages of infection;

[0028] Fig.13 This is a diagram showing the inhibition of the interaction between SARS-CoV-2S protein and hACE2 receptor by Ophiopogon japonicus saponin C;

[0029] Fig.14 This is a dose-response curve of the effect of Ophiopogon japonicus saponin C on the biological activity of receptor hACE2;

[0030] Fig.15 The inhibition of SARS-CoV-2S protein-mediated cell fusion by Ophiopogon japonicus saponin C (magnification = 10×10);

[0031] Fig.16 This is the relative fusion rate diagram under the action of broad-leaved Ophiopogon japonicus saponin C. DETAILED DESCRIPTION

[0032] The invention provides application of broad-leaved ophiopogon saponin C in preparing antiviral drugs.

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

[0034] In the present invention, the concentration of Ophiopogon japonicus saponin C used to inhibit H1N1 influenza virus is preferably 1.10 to 22 μM, more preferably 2.38 to 21 μM, and even more preferably 20 μM.

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

[0036] Preferably, Ophiopogon japonicus 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 form of the drug includes pills, tablets, powders, capsules, granules, powders, pellets, drops, sprays, injections, and suspensions.

[0038] The technical solutions provided by the present invention are 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 equipment

[0040] 1) Cells, strains and plasmids

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

[0042] MDCK cell line was obtained from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences;

[0043] A549 (human non-small cell lung cancer cells) were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences;

[0044] The luciferase reporter virus PR8-NS-Nluc was from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences;

[0045] The GFP-expressing plasmid pGIPZ-empty was purchased from OpenBiosystems;

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

[0047] SARS-CoV-2-S pseudovirions and VSV-G pseudovirions were from the School of Public Health, Lanzhou University.

[0048] 2) Compounds

[0049] Broadleaf Ophiopogon japonicus saponin C (HY-N5055) was purchased from MCE (MedChemExpress, USA) with a purity of more than 99%, dissolved in dimethyl sulfoxide (DMSO) solvent, and stored at a concentration of 10 mM.

[0050] 3) Experimental reagents

[0051] Table 1 Cell experiment reagents

[0052]

[0053] Table 2 Main experimental reagents

[0054]

[0055]

[0056] Table 3 Main experimental instruments

[0057]

[0058] Example 1: Radix Ophiopogonis Saponin C was administered throughout the course of treatment to study its inhibitory effect on influenza virus infection of MDCK cells and A549 cells

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

[0060] (1) MDCK cells in the logarithmic growth phase grown in DMEM medium containing 10% fetal bovine serum were cultured at a rate of 2×10 4 The cells were evenly inoculated at a density of 100 cells / well in a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator until the infection experiment was performed when the cells grew to about 80%.

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

[0062] (3) The supernatant was discarded, and the broadleaf ophiopogonin C was diluted into five gradients with serum-free DMEM medium containing TPCK-trypsin (1 μg / mL) to a final concentration of 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, respectively. The broadleaf ophiopogonin C dilutions of corresponding concentrations were then added to the well plate, and a virus control group without compound treatment was set up. The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 24 h.

[0063] (4) Then, luciferase was added to lyse the luminescence, and 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-added group / chemiluminescence value of the control group) × 100%, and GraphPad Prism 8.0 was used to draw a graph and calculate IC50.

[0064] Experimental results: Figure 1 As shown. Figure 1 It can be seen that the inhibition rate of broad-leaved ophiopogon saponin C on influenza virus infection in MDCK cells increases with the increase of concentration, and its IC50 is 1.10 μM.

[0065] In addition, the antiviral infection experiment of Ophiopogon japonicus saponin C was also carried out in A549 cells. 2 × 10 4 The cells were cultured until the cells grew to about 80%. Other operations were basically the same as the above MDCK cell infection experiment.

[0066] Experimental results: Figure 2 As shown. Figure 2 It can be seen that the inhibition rate of broad-leaved ophiopogon saponin C on influenza virus in A549 cells increases with the increase of concentration, and the IC50 is 2.38μM.

[0067] Example 2 Detection of the toxicity of Ophiopogon japonicus saponin C on MDCK cells and A549 cells by CCK8 method

[0068] MDCK cells in the logarithmic growth phase grown in DMEM medium containing 10% fetal bovine serum were cultured at a rate of 2×10 4 The density of cells / well was uniformly inoculated in a 96-well plate and cultured overnight in an incubator at 37°C with 5% CO2 until the cells grew to about 80%. The broad-leaved ophiopogonin C was diluted with DMEM diluent with a concentration of 100 μM in the first well, and diluted 2 times to 8 concentration gradients. The MDCK cells were incubated with different concentrations of broad-leaved ophiopogonin C at 37°C for 48 hours, and then CCK8 was used to detect cell activity (completely consistent with the drug administration process). The specific operation is as follows: 10 μL of CCK8 reagent was added to each well under light-proof conditions and incubated at 37°C for 1 hour. The wells with only DMEM culture solution containing 10% CCK8 reagent were used as blank controls. The absorbance of the cells at a wavelength of 450nm was detected by an enzyme marker. Cell activity calculation: Cell activity (%) = [OD (drug added) - OD (blank)] / [OD (control) - OD (blank)] × 100%.

[0069] Experimental results: Figure 3 As shown. Figure 3It can be seen that the median toxic concentration (CC50) of Ophiopogon japonicus saponin C in MDCK cells is 54.92 μM.

[0070] In addition, cytotoxicity assays were performed in A549 cells, with 2 × 10 4 cells, and cultured until the cells grew to about 80%, and diluted the broadleaf ophiopogonin C with DMEM diluent with a concentration of 100 μM in the first well, and diluted it 2-fold into 9 concentration gradients. Other operations were basically the same as the above MDCK cell toxicity test.

[0071] Experimental results: Figure 4 As shown. Figure 4 It can be seen that the median toxic concentration (CC50) of Ophiopogon japonicus saponin C on A549 cells is 25.04 μM.

[0072] The above results indicate that the effective inhibitory concentration of Ophiopogon japonicus saponin C on influenza virus infection is lower than its cytotoxic concentration, and it has good cytosafety.

[0073] Example 3 Determination of the Addition Time of Radix Ophiopogonis Saponin C

[0074] In order to clarify the effect of ophiopogonin C on influenza virus proliferation at different time points during the influenza replication cycle, a time-of-addition assay was performed. The specific method was as follows: MDCK cells in the logarithmic growth phase grown in DMEM medium containing 10% fetal bovine serum were cultured at 2×10 4 The density of cells / well was uniformly inoculated in a 96-well plate, and cultured overnight in an incubator containing 5% CO2 at 37°C until the cells grew to about 80% for infection experiments. The virus solution (MOI=0.05) was prepared with serum-free DMEM culture medium to infect the cells, and the well plate was placed in a cell culture incubator containing 5% CO2 at 37°C for 2 hours. The time point when the well plate was moved to the incubator was set to 0h.pi, and the cells were treated with DMSO or 10μM of broad-leaved ophiopogonin C at specified time intervals (0-2, 2-4, 4-6, 6-8, 8-10 hours and full administration, and the full administration was from the first 2 hours of virus infection of MDCK cells to 24 hours after infection), and then the cells were incubated with fresh culture medium until 24 hours after infection, and the luciferase activity in the cell lysate was detected, and the inhibition rate was calculated according to the chemiluminescence value of the control group to determine the action stage of broad-leaved ophiopogonin C in inhibiting influenza virus.

[0075] Experimental results: Figure 5 As shown, adding broad-leaved ophiopogonin C at 2-4h and 4-6h showed a higher inhibition rate, which was consistent with the inhibitory effect of full-course administration, indicating that broad-leaved ophiopogonin C plays a role in the early replication of the virus after it enters the cell.

[0076] Example 4: Radix Ophiopogonis Saponin C Affects the Nuclear-Cytoplasmic Shuttling of Influenza Virus vRNP

[0077] The shuttling of influenza virus vRNP complex between the nucleus and cytoplasm plays an important role in virus replication. In this study, at different time points after influenza virus infection, NP protein was labeled by indirect immunofluorescence to locate the position of vRNP in the cell, and then the effect of compound on the nuclear-cytoplasmic shuttling of influenza virus vRNP was observed by comparing the treatment with broadleaf ophiopogon saponin C or DMSO. The specific method is as follows:

[0078] (1) Prepare the slides: Take out a clean cover glass and soak it in 70% ethanol for 5 min. Then blow dry it on a sterile clean bench or wash it three times with cell culture grade PBS / 0.9% NaCl solution, and finally wash it once with cell culture medium.

[0079] (2) Cell Slides: Place a slide in a 12-well plate and culture MDCK cells in the logarithmic growth phase grown in DMEM medium containing 10% fetal bovine serum at a rate of 3×10 5 The density of cells / well was uniformly inoculated in a 12-well plate and cultured overnight in an incubator at 37°C with 5% CO2.

[0080] (3) Treatment: When the cells grew to about 80%, an infection experiment was performed, and a virus solution (MOI = 5) prepared with serum-free DMEM medium was added. After infection at 37°C for 1 hour, the supernatant was discarded, and broadleaf ophiopogonin C (10 μM) or DMSO diluted with serum-free DMEM medium containing TPCK-trypsin (1 μg / mL) was added. The supernatant was discarded at 2 h, 4 h, 6 h, and 8 h, respectively. The slides at different time points were taken out and washed 3 times with PBS (5 min / time, shaking or standing, the same below);

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

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

[0083] (6) Primary antibody incubation: Add QuickBlock TMImmunostaining: InfluenzaAvirusNP antibody (human Fc-tagged nanobody, 1:200) diluted in primary antibody diluent was incubated overnight at 4°C. The slides were washed 3 times with PBS;

[0084] (7) Secondary antibody incubation: Add QuickBlock TM Immunofluorescence staining: FITC-labeled goat anti-human IgG (H+L) diluted in secondary antibody diluent, incubate at room temperature in the dark for 1 hour. Wash the slides three times with PBS;

[0085] (8) Sealing: Add anti-fluorescence quenching sealing solution containing DAPI on the slide, remove the cell slide from the well plate, and place it upside down on the sealing medium for DAPI staining and sealing;

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

[0087] Experimental results: Figure 6 As shown by Figure 6 As shown in A, the vRNP complex in the DMSO group was mainly concentrated in the cytoplasm 2 hours after infection. At 4 hours after infection, the vRNP began to move to the nucleus. Until 8 hours after infection, the vRNP was distributed in both the cytoplasm and the nucleus. Figure 6 As shown in B, compared with the DMSO group, the vRNP complex was mostly concentrated in the cytoplasm after treatment with Ophiopogon japonicus saponin C, and the difference from the DMSO group was most obvious 8 hours after infection, indicating that Ophiopogon japonicus saponin C can inhibit the nuclear-cytoplasmic shuttling of vRNP after virus infection.

[0088] In summary, Ophiopogon japonicus saponin C can effectively inhibit influenza virus infection with an effective concentration of 1.25-20 μM and has low cytotoxicity. It is expected to be an effective anti-influenza virus infection drug with broad application prospects.

[0089] Example 5: Broadleaf Ophiopogonin C was administered throughout the course of administration to study its inhibitory effect on SARS-CoV-2 S protein pseudovirus-infected HEK293T / hACE2 cells and Vero E6 / hACE2 cells

[0090] HEK293T / hACE2 cells in the logarithmic growth phase grown in DMEM medium containing 10% fetal bovine serum were cultured at a rate of 2×10 4The density of cells / well was uniformly inoculated in a 96-well plate and cultured overnight in an incubator at 37°C with 5% CO2 until the cells grew to about 80% for infection experiments. Dilute the broadleaf ophiopogonin C into four concentration gradients of 20μM, 10μM, 5μM, and 1μM with DMEM diluent, mix the different concentrations of broadleaf ophiopogonin C with the SARS-CoV-2S protein pseudovirus solution, add an equal volume of DMEM diluent and a mixture of pseudovirus solution to the control group, incubate at 37°C for 48h, then discard the culture medium, then add luciferase to lyse and emit light, and use a multifunctional microplate reader 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-added group / chemiluminescence value of the control group) × 100%, and use GraphPad Prism 8.0 to draw a graph and calculate the half-maximal inhibitory concentration (Halfmaximal inhibitory Concentration, IC50).

[0091] Experimental results: Figure 7 As shown. Figure 7 It can be seen that the inhibition rate of broad-leaved ophiopogon saponin C on SARS-CoV-2 pseudovirus infection in HEK293T / hACE2 cells increases with the increase of concentration, and its half inhibitory concentration (IC50) = 1.50 μM.

[0092] In addition, infection experiments were performed in Vero E6 / hACE2 cells. 1.5 × 10 4 The cells were cultured until the cells grew to about 80%. Other operations were basically the same as the above HEK293T / hACE2 cell infection experiment.

[0093] Experimental results: Figure 8 As shown. Figure 8 It can be seen that the inhibition rate of broad-leaved ophiopogon saponin C on SARS-CoV-2 pseudovirus in Vero E6 / hACE2 cells increases with the increase of concentration, and its half inhibitory concentration (IC50) = 7.18 μM.

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

[0095] HEK293T / hACE2 cells in the logarithmic growth phase grown in DMEM medium containing 10% fetal bovine serum were cultured at a rate of 2×10 4The density of cells / well was uniformly inoculated in a 96-well plate, and cultured overnight in an incubator at 37°C with 5% CO2. When the cell confluence reached about 80%, the broad-leaved ophiopogonin C was diluted with DMEM diluent with a concentration of 100 μM in the first well, and diluted 2-fold to 8 concentration gradients. HEK293T / hACE2 cells were incubated with different concentrations of broad-leaved ophiopogonin C at 37°C for 48 hours, and then CCK8 was used to detect cell activity (completely consistent with the drug administration process). The specific operation is as follows: 10 μL of CCK8 reagent was added to each well and incubated at 37°C in the dark for 2 hours. The wells with only DMEM culture solution containing 10% CCK8 reagent were used as blank controls. The absorbance of the cells at a wavelength of 450nm was detected by an enzyme marker. Cell activity calculation: Cell activity (%) = [OD (drug added) - OD (blank)] / [OD (control) - OD (blank)] × 100%.

[0096] Experimental results: Fig. 9 As shown. Fig. 9 It can be seen that the median toxic concentration (CC50) of Ophiopogon japonicus saponin C in HEK293T / hACE2 cells is 69.01 μM.

[0097] In addition, cytotoxicity assays were performed in Vero E6 / hACE2 cells, with 1.5 × 10 4 cells, and cultured until the cells grew to about 80%, and diluted the broad-leaved ophiopogonin C with DMEM diluent with a first-well concentration of 200 μM, 2-fold dilution into 8 concentration gradients, and other operations were basically the same as the above HEK293T / hACE2 cell toxicity assay.

[0098] Experimental results: Fig.10 As shown. Fig.10 It can be seen that the median toxic concentration (CC50) of Ophiopogon japonicus saponin C on Vero E6 / hACE2 cells is 106.4 μM.

[0099] The above results show that the effective inhibitory concentration of broad-leaved Ophiopogon japonicus saponin C on SARS-COV-2 pseudovirus infection is much lower than its cytotoxic concentration, and has good cell safety.

[0100] Example 7: Broadleaf Radix Ophiopogonis Saponin C has specificity in inhibiting the invasion of SARS-COV-2 pseudovirus

[0101] In order to identify whether broadleaf ophiopogon saponin C can inhibit SARS-CoV-2 infection, the inhibitory effect of broadleaf ophiopogon saponin C on SARS-CoV-2 pseudovirus was tested. The specific method is as follows:

[0102] (1) HEK293T / hACE2 cells in the logarithmic growth phase grown in DMEM medium containing 10% fetal bovine serum were cultured at a rate of 2×10 4 The cells were uniformly inoculated at a density of 100 cells / well in a 96-well plate and placed in a 37°C incubator containing 5% CO2 overnight. The experiment was performed when the cells grew to about 80%.

[0103] (2) Dilute the broadleaf ophiopogonin C with DMEM diluent to a final concentration of 20 μM, 10 μM, 5 μM, and 1 μM into 4 gradients, then mix the corresponding concentrations of broadleaf ophiopogonin C with the SARS-CoV-2 pseudovirus solution to co-infect the target cells, and add an equal volume of the mixture of DMEM diluent and pseudovirus solution to the control wells;

[0104] (3) VSV-G pseudovirus was used as a control for infection experiments to verify the specificity of Ophiopogon japonicus saponin C against SARS-CoV-2. The experimental procedures for using VSV-G pseudovirus as a control were the same as above.

[0105] (4) After incubation for 48 h in a 37°C, 5% CO2 incubator, the cells were taken out and a volume of luciferase equal to that of the supernatant was added to the wells in a dark environment to detect the chemiluminescence value using a multifunctional microplate reader. The relative infection rate (%) was calculated based on the control group = (chemiluminescence value of the drug-treated group / chemiluminescence value of the control group) × 100%.

[0106] Results: Fig.11 As shown in the data, the inhibitory effect of broad-leaved ophiopogon saponin C on VSV-G pseudovirus was weak, far lower than its activity against SARS-CoV-2. Broad-leaved ophiopogon saponin C had no antiviral activity against VSV-G pseudovirus at the maximum concentration of 20 μM, and even the relative infection rate was slightly increased. This result indicates that broad-leaved ophiopogon saponin C is specific in resisting SARS-CoV-2 infection.

[0107] Example 8: Broadleaf Ophiopogon japonicus saponin C inhibits SARS-CoV-2 pseudovirus invasion at an early stage

[0108] In order to clarify the stage at which Ophiopogon japonicus saponin C exerts its anti-SARS-CoV-2 infection effect, 293T-hACE2 cells were treated with 10 μM of the compound before (-1 h), during (0 h), and after (2 h) pseudovirus infection. The luciferase activity in the cell lysate was detected 48 h later, and the inhibition rate was calculated based on the chemiluminescence value of the control group.

[0109] Results: Fig.12As shown, the broadleaf ophiopogonin C showed the highest inhibition rate when added before infection, that is, the drug was incubated with cells for 1 hour before adding SARS-CoV-2 pseudovirus, which was better than the inhibition rate when added during infection. The inhibition rate of adding the drug 2 hours after pseudovirus infection was lower than that before and during infection. This result shows 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 ELISA method to detect the effect of broadleaf ophiopogon saponin C on the binding of S protein and hACE2

[0111] Using CoviDrop TM The SARS-CoV-2 Spike-ACE 2 Binding Inhibitor Screening Rapid Kit (D-1004) was used for the experiment. 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 at a ratio of 1:2000 to the working concentration; dilute ACE2 protein and detection buffer at a ratio of 1:1000 to prepare ACE2 working solution; use the detection buffer to dilute the positive control into a strong positive control solution (1 mg / mL) and a weak positive control solution (100 μg / mL);

[0113] (2) Adding samples of Ophiopogon japonicus saponin C: set up a control group according to Table 4 and add samples;

[0114] Table 4 Sample loading table for SARS-CoV-2 Spike-ACE 2 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) Adding ACE2 working solution: After incubation 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) Washing: Discard the solution in the wells, add 150 μL of 1× washing solution to each well using a dispenser, shake the plate for 45 seconds, and repeat 3 times;

[0120] (7) Adding binding test solution: Use a dispenser to add 50 μL of binding test solution to each well;

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

[0122] (9) Washing: Discard the solution in the wells, add 150 μL of 1× washing solution to each well using a dispenser, shake the plate for 45 seconds, and repeat 5 times;

[0123] (10) Adding developer: Use a spray gun to add 100 μL of developer to each well;

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

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

[0126] (13) Determination of OD value: The OD value was measured using a multifunctional microplate reader (Tecan infinite 200, TECAN), with a measurement wavelength of 450 nm and a reference wavelength of 655 nm;

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

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

[0129] Example 10: Broadleaf Ophiopogon japonicus saponin C has no effect on the biological function of hACE2 receptor

[0130] In order to explore the potential effect of Ophiopogon japonicus saponin C on hACE2 receptor, the inhibitory effect of the compound on hACE2 protease activity was determined using the Bio-Time Angiotensin Converting Enzyme 2 (ACE2) Inhibitor Screening Kit (P0320S). The specific steps are as follows:

[0131] (1) Sample preparation: Use Assay Buffer to prepare a concentration gradient of Ophiopogon japonicus saponin C with final concentrations of 500 μM, 100 μM, 20 μM, 4 μM, 0.8 μM and 0.16 μM;

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

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

[0134] (4) Sample addition: Add the detection reagent and sample to the 96-well black plate in sequence according to Table 5 below and mix well. Then use a spray gun to add 2 μL of Substrate to each well and mix quickly.

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

[0136]

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

[0138] (6) Determination of fluorescence value: The multifunctional microplate reader was programmed to have an excitation wavelength of 325 nm and an emission wavelength of 393 nm;

[0139] (7) Calculate the relative inhibition rate: Calculate the average value 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) Calculation of IC50: GraphPadPrism 8.0 was used to plot and calculate the IC50 of Ophiopogon japonicus saponin C.

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

[0142] Example 11 Effect of Radix Ophiopogonis Saponin C on Cell Fusion Mediated by S Protein of SARS-CoV-2

[0143] Effector cells expressing SARS-CoV-2 S protein and GFP (HEK293T / SARS-CoV-2S / GFP) and target cells expressing hACE2 receptors on the cell surface (HEK293T / hACE2) were co-cultured to allow the S protein to mediate cell-to-cell fusion by recognizing hACE2. When the effector cells and target cells fuse to form syncytia, the fused cells are usually 2 times larger than the unfused cells, and because GFP diffuses from an effector cell to the target cell, the fluorescence intensity of the fused cells will weaken, thereby detecting the effect of broadleaf ophiopogonin C on cell fusion. The specific method is as follows:

[0144] (1) HEK293T cells were cultured at 4×10 4 The cells were inoculated at a density of 100 / mL in a 6-well plate and cultured overnight in a cell culture incubator at 37°C with 5% CO2.

[0145] (2) When the cell confluence reached about 80%, the cell supernatant was discarded and 2 mL of preheated DMEM complete medium was added to each well. In the experimental group, DNA (pVAX1-nCoVS and pGIPZ-empty) was mixed with lipo8000 transfection reagent at a mass volume ratio of 1:2 and co-transfected into HEK293T cells. In the negative control group, only pGIPZ-empty was transfected in the same way.

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

[0147] (4) 24 h after transfection, HEK293T-hACE2 cells were seeded in a 96-well transparent cell culture plate and cultured in a cell culture incubator at 37°C with 5% CO2;

[0148] (5) After 48 hours of transfection, the 6-well plate was removed, the supernatant was discarded, and the effector cells (HEK293T / SARS-CoV-2S / GFP and HEK293T / GFP cells) were resuspended in DMEM complete medium and counted. Broadleaf Ophiopogonin C was diluted with DMEM diluent to three concentration gradients of 5 μM, 10 μM, and 20 μM, and then HEK293T / SARS-CoV-2S / GFP effector cells were incubated with different concentrations of Broadleaf Ophiopogonin C for 30 minutes, and then 2×10 4 Add 100 μL of cell-drug mixture to the target cells at a concentration of cells / mL, add an equal volume of HEK293T / SARS-CoV-2S / GFP effector cell suspension to the positive control group, and add an equal volume of HEK293T / GFP effector cell suspension to the negative control group;

[0149] (6) After 6 h of co-culture at 37°C, 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. Cell fusion rate (%) = (EN) / (PN) × 100%; E is the ratio of the experimental combination cell body to the total number of cells, P is the ratio of the positive control combination cell body to the total number of cells, and N is the ratio of the negative control combination cell body to the total number of cells.

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

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

[0152] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of broad-leaved ophiopogon saponin C in the preparation of antiviral drugs.

2. The use according to claim 1, characterized in that The virus is H1N1 influenza virus or SARS-CoV-2.

3. The use according to claim 2, characterized in that: The concentration of broad-leaved ophiopogon saponin C in inhibiting H1N1 influenza virus is 1.10-22 μM.

4. The use according to claim 2, characterized in that: The concentration of broad-leaved ophiopogon saponin C to inhibit SARS-CoV-2 is 1 to 22 μM.

5. The use according to claim 1, characterized in that: Broadleaf Ophiopogon japonicus saponin C can bind to the S protein of SARS-CoV-2.

6. The use according to any one of claims 1 to 5, 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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