Application of 4-hydroxyderrisin in preparation of anti-influenza virus drugs

Abstract

The invention belongs to the field of new application of medicines, and particularly relates to application of 4-hydroxyderrisin in preparation of anti-influenza virus medicines. It is found for the first time that 4-hydroxyderrisin can significantly inhibit replication and internalization of influenza A virus H1N1 and has significant anti-influenza virus activity, the median inhibitory concentrations of 4-hydroxyderrisin in MDCK cells and A549 cells are 0.60 M and 0.23 M respectively, and the 4-hydroxyderrisin is low in cytotoxicity and high in safety. Experiments show that 4-hydroxyderrisin plays a role in the early stage of a virus life replication cycle, and plays an antiviral role by inhibiting virus internalization without affecting virus adsorption and neuraminidase activity. The medicine can be prepared into a solid or solution dosage form, is used for preventing and treating influenza, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of new uses of pharmaceuticals, and more specifically, relates to the use of 4-hydroxydrycin in the preparation of antiviral drugs for influenza. Background Technology

[0002] Influenza is a common respiratory infection that typically occurs in winter, causing acute fever, often accompanied by muscle pain, headache, and cough. During epidemics, the incidence of complications and mortality rates after influenza infection increase significantly. The pathogen, the influenza virus, belongs to the Orthomyxoviridae family and is a single-stranded, negative-sense RNA virus, approximately 13.6 kb in length. Based on the antigenicity of its nucleoprotein and matrix protein, influenza viruses can be classified into types A, B, C, and D. Type A influenza viruses have a wide host range, are more prone to antigenic mutation than other influenza viruses, and exhibit greater variability. Therefore, it is not only the most common and deadliest type of influenza virus but also the primary cause of seasonal or pandemic influenza. Among type A influenza viruses, H1N1 is one of the major serotypes. Since the 1918 "Spanish flu" outbreak, this virus has caused 50 million deaths this century. The World Health Organization estimates that influenza epidemics cause approximately 4 million severe cases and about 500,000 deaths annually.

[0003] Currently, influenza is primarily treated with antiviral drugs. Besides broad-spectrum antiviral drugs, three classes of antiviral drugs are approved for influenza prevention and treatment, targeting the influenza virus matrix protein 2 (M2) ion channel, neuraminidase (NA), and polymerase cap-dependent endonuclease (CEN), respectively. However, due to the high variability of the influenza virus genome, drug resistance is a significant challenge in drug treatment. M2 ion channel inhibitors are no longer recommended for influenza prevention and treatment. NA inhibitors, represented by oseltamivir, are the most commonly used influenza treatment drugs. Spontaneously acquired or post-drug exposure NA protein H275Y mutations can lead to oseltamivir resistance in influenza viruses. Furthermore, non-H275Y NA substitutions can also reduce oseltamivir sensitivity. Baloxavir is a representative CEN inhibitor. Some studies have shown that approximately 10% of patients develop a specific mutation (PA / I38X) after treatment, which is associated with higher viral loads and prolonged clinical symptoms.

[0004] For millennia, natural phytochemicals have been used to prevent and treat various diseases. Natural phytochemicals are mainly classified into four categories: alkaloids, phenolic derivatives, terpenes, and other metabolites. Studies have shown that many chemicals in these four categories possess broad-spectrum antibacterial and antiviral activities. Flavonoids belong to the phenolic derivative class and are also the largest class of secondary metabolites in plants, widely found in various plants and berries in nature, totaling approximately 4,000 species. Celery, parsley, red peppers, chamomile, mint, and ginkgo leaves are major sources of flavonoids. Past studies have found that various flavonoids, such as Rhodiola rosea isolate, elderberry fruit extract, and honeysuckle extract, can inhibit influenza virus activity in vitro and in vivo. 4-Hydroxyderricin is the main active ingredient of Angelica keiskei (Ashitaba), exhibiting activity that inhibits glucose uptake, which has been verified in mice. However, there is currently no research on the inhibitory effect of 4-hydroxyderricin on influenza viruses, making the exploration of the effects of 4-hydroxyderricin on influenza viruses a promising area for future research. To further expand the application scope of 4-hydroxydrycin, further research is needed on its other pharmaceutical uses. Summary of the Invention

[0005] The purpose of this invention is to provide the use of 4-hydroxydrycin in the preparation of anti-influenza virus drugs.

[0006] This invention provides the use of 4-hydroxydrycin in the preparation of anti-influenza virus drugs.

[0007] This invention utilizes the PR8-NS-Nluc luciferase reporter virus of influenza A (H1N1) to quantitatively evaluate influenza virus replication at the cellular level by detecting the expression level of the tag protein luciferase gene NanoLuc Luciferase (Nluc) in infected cells. To eliminate false positive results due to low cell viability caused by the potential cytotoxicity of the compound, the cytotoxicity of the compound was further detected using the CCK8 assay. Crystal violet staining was used to observe the cytopathic effects after viral infection and the improvement effect of the compound treatment on cytopathic effects under a microscope. qRT-PCR was used to measure the levels of vRNA, cRNA, and mRNA in virus-infected cells under different treatments to evaluate the effect of the compound on influenza virus RNA transcription. Western blotting was used to detect the expression of influenza virus NP protein after compound treatment to evaluate influenza virus replication. By detecting the inhibition rate of the compound against influenza virus under different administration methods, the stage of the compound's inhibitory effect on influenza virus was preliminarily determined. Time-series experiments were used to evaluate the effect of the compound administered at different time points on influenza virus replication, clarifying the specific stage at which the compound exerts its effect within the influenza replication cycle. The effects of the compound on the adsorption and internalization phases of influenza virus in cells were evaluated using adsorption and internalization experiments. The effect of the compound on neuraminidase activity was detected using a neuraminidase inhibitor assay kit. Based on these experiments, this invention is the first to discover that 4-hydroxydryline can improve cytopathic effects caused by H1N1 virus, reduce the expression levels of H1N1 virus mRNA and NP protein in cells, and inhibit viral replication and internalization. These experiments demonstrate the effectiveness of the single-component composition of this invention. 4-hydroxydryline can also be combined with other antiviral drugs, such as honeysuckle, and the compound composition may exhibit synergistic effects (i.e., the effect is better than the sum of the effects of using either drug alone), additive effects, or advantages such as reducing drug resistance.

[0008] Furthermore, the influenza virus includes H1N1.

[0009] Furthermore, the antiviral drug is used to inhibit influenza virus replication and internalization.

[0010] The present invention also provides an antiviral drug composition comprising 4-hydroxydrycin or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers and / or excipients.

[0011] Furthermore, the pharmaceutical composition also contains one or more other antiviral drugs.

[0012] Furthermore, the other antiviral drugs are compounds with antiviral activity and / or extracts of traditional Chinese medicine, and / or compositions of traditional Chinese medicine with antiviral effects.

[0013] Furthermore, the pharmaceutical composition is administered orally or by injection.

[0014] Furthermore, the formulations of the pharmaceutical composition include tablets, capsules, granules, dispersants, oral liquids, injections, sprays, and drops.

[0015] The beneficial effects of this invention are as follows: 4-Hydroxydroxycin significantly inhibited H1N1 virus infection. In MDCK cells, the half-maximal inhibitory concentration (IC50) of 4-hydroxydroxycin against H1N1 virus was [value missing]. 50 At 0.60 µM, 4-hydroxydrycin showed an IC50 inhibitory effect against H1N1 virus on A549 cells. 50 The concentration was 0.23 µM. 4-Hydroxydroxynine inhibits H1N1 virus replication by suppressing the early internalization phase of the virus. Attached Figure Description

[0016] Figure 1 Figure 1 shows the inhibition rate and cytotoxicity of 4-hydroxydrycin against influenza virus-infected MDCK cells.

[0017] Figure 2 Figure 1 shows the inhibition rate and cytotoxicity of 4-hydroxydrycin against influenza virus-infected A549 cells.

[0018] Figure 3 Cytopathic effect of 4-hydroxydrycin on influenza virus infection MDCK.

[0019] Figure 4 The graph shows the inhibitory effect of 4-hydroxydrycin on influenza virus mRNA synthesis at different treatment times. In the graph, A is the statistical graph of the inhibitory effect on mRNA synthesis after 4 hours of treatment, and B is the statistical graph of the inhibitory effect on mRNA synthesis after 8 hours of treatment.

[0020] Figure 5 The diagram shows the effect of 4-hydroxydrycin on NP protein expression in MDCK cells infected with influenza virus. In the diagram, A is the immunoblotting detection of NP protein and B is the quantitative analysis of NP protein.

[0021] Figure 6 The graph shows the inhibitory effect of 4-hydroxydracheline on influenza virus infection of MDCK cells under different administration methods. In the graph, A is a schematic diagram of the administration method of 4-hydroxydracheline, and B is a graph showing the inhibition rate of 4-hydroxydracheline on influenza virus infection of MDCK cells under different administration methods.

[0022] Figure 7 The graph shows the inhibitory effect of 4-hydroxydridine on influenza virus replication when added at different time periods. In the graph, A is a schematic diagram of the time of 4-hydroxydridine addition, and B is a graph showing the inhibition rate of 4-hydroxydridine added at different time periods on influenza virus infection of MDCK cells.

[0023] Figure 8 The diagram shows the effect of 4-hydroxydresisine on the adsorption and internalization of influenza virus. In the diagram, A shows the inhibitory effect of 4-hydroxydresisine on the virus adsorption stage, and B shows the inhibitory effect of 4-hydroxydresisine on the virus internalization stage.

[0024] Figure 9 The diagram shows the inhibitory effect of 4-hydroxydryline on neuraminidase activity. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0026] Example 1: 4-Hydroxydroxycin was administered throughout the course of treatment to study its inhibitory effect on H1N1-infected MDCK cells and H1N1-infected A549 cells.

[0027] The luciferase reporter virus PR8-NS-Nluc (H1N1) used in this experiment was purchased from the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. Virus amplification was performed using SPF-grade 11-day-old chicken embryos. The original virus solution was diluted 10... 5 After doubling, inject 100 μl into the allantoic cavity of chicken embryos. Incubate at 37°C for 48 hours, collect the allantoic fluid after virus amplification, filter, centrifuge, aliquot and store in an ultra-low temperature freezer at -80°C for later use.

[0028] MDCK cells in logarithmic growth phase, grown in DMEM medium containing 10% fetal bovine serum, were cultured at a rate of 2 × 10⁻⁶ cells / mL. 4Cells were evenly seeded at a density of cells / well in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator until the cell confluence reached approximately 80%. Infection experiments were then performed. The required viral load was calculated based on a multiplicity of infection (MOI) of 0.01, and a viral dilution was prepared using serum-free DMEM medium. 50 μL / well of H1N1 virus dilution with an MOI of 0.01 was added to each cell well as the virus control group. 50 μL / well of H1N1 virus dilution with MOI of 0.01 containing 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, 0.78 μM, 0.39 μM, and 0.19 μM 4-hydroxydrycin were added to each cell well as the compound treatment groups. Both groups were co-cultured at 37°C for 24 h. Then, an equal volume of Nano-Glo® HiBiT lysis reagent was added, and the mixture was thoroughly mixed before the reaction was carried out. The chemiluminescence value (CL value) was then detected using a multi-mode microplate reader. The inhibition rate was calculated compared to the virus control group: Inhibition rate (%) = [1 - CL (compound treatment group) / CL (virus control group)] × 100%. Graphs were plotted using GraphPad Prism 8.0, and the half-maximal inhibitory concentration (IC50) was calculated based on the inhibition rate. 50 ).

[0029] Experimental results: such as Figure 1 As shown, the inhibitory rate of 4-hydroxydrycin against H1N1 virus infection in MDCK cells increased with increasing concentration, and its IC50 value increased. 50 =0.60µM.

[0030] In addition, an experiment was conducted in A549 cells to investigate the effects of 4-hydroxydrycin on H1N1 virus infection, following the same experimental procedures as described above.

[0031] Experimental results: such as Figure 2 As shown. By Figure 2 It can be seen that the inhibitory rate of 4-hydroxydrycin against H1N1 virus in A549 cells increases with increasing concentration, and the IC50 value is [not specified]. 50 It is 0.23µM.

[0032] Example 2: CCK8 assay for the toxicity of 4-hydroxydrycin to MDCK and A549 cells.

[0033] MDCK cells in logarithmic growth phase, grown in DMEM medium containing 10% fetal bovine serum, were cultured at a ratio of 2 × 10⁻⁶ cells / mL. 4Cells were evenly seeded at a density of cells / well in 96-well plates and cultured overnight at 37°C in a 5% CO2 incubator until cell confluence reached approximately 80%. 50 μL / well of 4-hydroxydryline dilutions (containing no 4-hydroxydryline or final concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, 0.78 μM, and 0.39 μM) were added to each well as the cell control and compound treatment groups, respectively, and incubated at 37°C for 48 h. After 48 h, the cell supernatant was discarded, and 100 μL of a pre-prepared assay solution (DMEM medium to CCK8 reagent at a volume ratio of 9:1) was added in the dark. The cells were then incubated at 37°C for another 2 h. Wells containing only DMEM medium with 10% CCK8 reagent served as blank controls. Cell viability was detected using a multi-functional microplate reader. The detection wavelength was set to 450 nm, and the reference wavelength was 630 nm. Absorbance values ​​(OD values) were measured. Cell viability (%) = [OD(compound group) - OD(blank control group)] / [OD(cell control group) - OD(blank control group)] × 100%. GraphPad Prism 8.0 was used for plotting, and the half-maximal toxicity concentration (CC50) was calculated based on the cell viability. 50 ).

[0034] Experimental results: such as Figure 1 As shown, 4-hydroxydrycin CCC on MDCK cells 50 =34.39µM.

[0035] In addition, cytotoxicity tests were performed in A549 cells, following the same experimental procedures as described above.

[0036] Experimental results: such as Figure 2 As shown, 4-hydroxydrycin CCC on A549 cells 50 =37.43µM.

[0037] Example 3: 4-Hydroxydroxycinne can improve cytopathic effects caused by H1N1 virus.

[0038] MDCK cells in logarithmic growth phase, grown in DMEM medium containing 10% fetal bovine serum, were cultured at a concentration of 1.5 × 10⁻⁶ cells / mL. 4Cells were evenly seeded at a density of cells / well in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator until cell confluence reached approximately 80%. 50 μL of serum-free DMEM medium (diluent) was added to each well as a blank control group. All other treatment groups received H1N1 virus dilution with an MOI of 0.05. The virus control group received an additional dilution without any compounds, the positive control group received an additional dilution containing 1 nM baloxavir, and the compound treatment groups received additional dilutions containing 1 μM, 5 μM, and 10 μM 4-hydroxydrycin. All treatment groups were incubated at 37°C for 24 h. After 24 h, the supernatant was discarded, and cells were fixed with 4% paraformaldehyde. After 2 h, the fixative was discarded, and cells were stained with 1% crystal violet for 4 h. After 4 h, the staining solution was discarded, excess stain was washed off, and the wells were observed and photographed under an optical microscope.

[0039] Experimental results: such as Figure 3 As shown. By Figure 3 It is known that 4-hydroxydrycin can improve MDCK cell cytopathic effects caused by H1N1 virus, and the improvement effect increases with increasing compound concentration.

[0040] Example 4: 4-Hydroxydroxycin significantly reduced H1N1 virus mRNA expression.

[0041] MDCK cells in logarithmic growth phase, grown in DMEM medium containing 10% fetal bovine serum, were cultured at a rate of 6 × 10⁻⁶. 4 Cells were evenly seeded at a density of [number] cells / well in 24-well plates and incubated overnight at 37°C in a 5% CO2 incubator until approximately 80% confluence was achieved, at which point infection experiments were performed. Cells were infected with H1N1 virus dilution at MOI=0.01, with 250 μL of virus dilution added to each well. The virus control group received 250 μL of virus dilution along with an equal volume of dilutions containing neither virus nor the compound. The positive control group received an equal volume of baloxavir at a final concentration of 1 nM after adding the virus solution, while the compound groups received equal volumes of 4-hydroxydrycin dilutions at final concentrations of 5, 10, and 15 μM. After co-incubation at 37°C for 4 h and 8 h, RNA samples were extracted, and reverse transcription was performed using a kit. The transcriptional level of H1N1 virus mRNA under different treatments was detected by qRT-PCR.

[0042] Experimental results: such as Figure 4 As shown. By Figure 4 It is known that 4-hydroxydridine can significantly reduce the transcription level of viral mRNA, and the viral mRNA copy number decreases with increasing 4-hydroxydridine concentration.

[0043] Example 5: 4-Hydroxydroxycinnamic acid can reduce the expression of NP protein after H1N1 virus infection.

[0044] MDCK cells in logarithmic growth phase, grown in DMEM medium containing 10% fetal bovine serum, were cultured at a ratio of 8 × 10⁸ cells / year. 4 Cells were evenly seeded at a density of cells / well in 6-well plates and incubated overnight at 37°C in a 5% CO2 incubator until the cells reached approximately 80% confluence. The cell control group received 1 mL of diluent without H1N1 virus and the compound; the virus control group received 1 mL of diluent containing H1N1 virus (MOI=0.01); the positive control group received 1 mL of H1N1 virus diluent containing 1 nM baloxavir (MOI=0.01); and the compound group received 1 mL of H1N1 virus diluent containing 2.5 μM, 5 μM, and 10 μM 4-hydroxydrycin (MOI=0.01). All cells were incubated at 37°C for 24 h. After 24 h, cell samples were collected and lysed to obtain protein-containing supernatants. Western blotting (WB) was performed using the protein supernatants to detect the effect of the compounds on the expression of viral NP proteins. Images were then analyzed using ImageJ software after imaging with a protein gel imaging system.

[0045] Experimental results: such as Figure 5 As shown. By Figure 5 As shown in B, 4-hydroxydrycin can reduce the expression level of NP protein after H1N1 virus infection, and this effect is dose-dependent.

[0046] Example 6: 4-Hydroxydroxycin can exert anti-H1N1 virus effects under multiple administration modes.

[0047] MDCK cells in logarithmic growth phase, grown in DMEM medium containing 10% fetal bovine serum, were cultured at a rate of 2 × 10⁻⁶ cells / mL. 4The cells were evenly seeded at a density of cells / well in 96-well plates and cultured overnight at 37°C in a 5% CO2 incubator until the cell confluence reached about 80% for infection experiments. The administration methods can be divided into: (1) Virus pretreatment: 50 μL of H1N1 virus dilution (MOI=0.01) was mixed with an equal volume of 4-hydroxydrycin dilution and incubated at 4°C for 2 h. After 2 h, the mixture was added to MDCK cells at 100 μL / well and incubated with the cells at 37°C. After 2 h, the supernatant was discarded and the cells were washed. The cells were then cultured in a dilution free of virus and compound for 24 h; (2) Cell pretreatment: 4-hydroxydrycin dilution was added to the cell wells and incubated at 4°C for 2 h. After 2 hours, the supernatant was discarded and washed, and 50 μL of H1N1 virus dilution was added. The cells were incubated together at 37°C. After 2 hours, the supernatant was discarded and washed, and the cells were incubated with dilution containing no virus or compound for 24 hours. (3) Co-treatment: 50 μL of H1N1 virus dilution and 50 μL of 4-hydroxydrycin dilution were added together to the cell wells and incubated at 37°C. After 2 hours, the supernatant was discarded and washed, and the cells were incubated with dilution containing no virus or compound for 24 hours. (4) Post-infection administration: 50 μL of H1N1 virus dilution was added to the cell wells and incubated at 37°C. After 2 hours, the supernatant was discarded and washed, and the cells were incubated with dilution containing 4-hydroxydrycin for 24 hours. (5) Full-process administration: The cells were incubated with dilution containing 4-hydroxydrycin from -2 to 24 hours. 50 μL / well of H1N1 virus dilution was only incubated with the cells and compound from 0 to 2 hours. The final concentration of 4-hydroxydroxydroxycin in the incubation system was 10 μM in all five groups above. After 24 h, the CL value was detected using Nano-Glo® HiBiT lysis reagent, and the H1N1 virus inhibition rate of 4-hydroxydroxydroxycin under different routes of administration was calculated.

[0048] Experimental results: such as Figure 6 As shown. By Figure 6 As shown in B, 4-hydroxydriceline exhibits high inhibition rates against H1N1 virus under different administration routes. The inhibitory effect is weaker under cell pretreatment, but the inhibition rates of 4-hydroxydriceline against H1N1 virus are very close to those of full-course administration during virus pretreatment, co-treatment, and post-infection treatment.

[0049] Example 7: The role of 4-hydroxydrycin in different stages of H1N1 virus replication.

[0050] MDCK cells in logarithmic growth phase grown in DMEM medium containing 10% fetal bovine serum were cultured at a ratio of 2 × 10⁻⁶ cells / mL. 4Cells were evenly seeded at a density of cells / well in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator until cell confluence reached approximately 80%. H1N1 virus was diluted to 0.01 MOI to infect cells, with 50 μL of virus solution added to each well. The time point for virus addition to cells was set to 0 h. 4-Hydroxydroxycinnamate was added at specified time intervals during virus infection (-2~0 h, 0~2 h, 2~4 h, 4~6 h, 6~8 h, and the entire course of administration, i.e., -2~24 h) to a final concentration of 10 µM. Cells were collected uniformly 24 h post-infection, and viral replication was detected using the Nano-Glo® HiBiT lysis reagent.

[0051] Experimental results: such as Figure 7 As shown. By Figure 7 As shown in Figure B, the addition of 4-hydroxydrone between 0 and 2 hours exhibited a high inhibition rate consistent with the overall timeframe. Furthermore, the highest inhibition rate was observed when 4-hydroxydrone was added between -2 and 0 hours. The inhibitory effect of 4-hydroxydrone gradually decreased with increasing time of addition, and when added between 6 and 8 hours after infection, it showed virtually no inhibition of H1N1 virus replication. This indicates that 4-hydroxydrone plays a role in the early stages of the viral replication cycle.

[0052] Example 8: 4-Hydroxydroxycin does not affect the adsorption of H1N1 virus, but significantly inhibits its internalization.

[0053] MDCK cells in logarithmic growth phase, grown in DMEM medium containing 10% fetal bovine serum, were cultured at a rate of 2 × 10⁻⁶ cells / mL. 4 Cells were evenly seeded at a density of cells / well in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator until approximately 80% confluence was achieved, at which point infection experiments were performed. The adsorption experiment was performed as follows: the culture medium in the wells was discarded, and a diluent containing or without 4-hydroxydryline was added. After incubation for 2 hours, 50 μL of H1N1 virus diluent (MOI=5) was added to the wells, and co-incubated at 4°C. After 1 hour, the cells were washed 8 times with pre-chilled sterile PBS to remove unadsorbed virus particles, and immediately detected using Nano-Glo® HiBiT lysis reagent. The internalization experiment was performed as follows: the culture medium in the wells was discarded, and a diluent containing or without 4-hydroxydryline was added. After incubation for 1 hour, the plate was placed in an ice box, and 50 μL of H1N1 virus diluent (MOI=5) was added. Cells were infected at 4°C for 1 hour, followed by incubation at 37°C for 1 hour to allow virus internalization. One hour later, the cells were washed three times with sterile PBS at pH 1.3 and immediately detected using Nano-Glo® HiBiT lysis reagent.

[0054] Experimental results: such as Figure 8 As shown. By Figure 8 As shown in A, 4-hydroxydryline does not affect the adsorption of H1N1 virus to cells; Figure 8 As shown in B, 4-hydroxydrycin can reduce the internalization of H1N1 virus, and the inhibitory effect is enhanced with increasing concentration.

[0055] Example 9: 4-Hydroxydroxycin does not inhibit NA activity.

[0056] The activity of compounds against neuraminidase (NA) was detected using a neuraminidase (NA) inhibitor screening kit. Following the manufacturer's instructions, NA detection buffer, NA, and the sample to be tested were added to a 96-well black plate, mixed thoroughly, and then NA fluorescent substrate was added and incubated at 37°C for 30 min. The positive control group was treated with 10 μL of 20 μM oseltamivir, the compound treatment groups were treated with equal volumes of 25, 50, 100, and 200 μM 4-hydroxydrycin, and the blank control group was treated with an equal volume of sample dilution buffer. A multi-mode microplate reader was set to an excitation wavelength of 322 nm and an emission wavelength of 450 nm to detect fluorescence intensity (FL). Inhibition rate (%) = [1 - FL (compound treatment group) / CL (blank control group)] × 100%.

[0057] Experimental results: such as Figure 9 As shown. By Figure 9 It can be seen that 4-hydroxydrycin does not affect the activity of NA.

[0058] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0059] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0060] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of 4-hydroxyderridin in the preparation of an anti-influenza virus drug.

2. Use according to claim 1, characterized in that, The influenza virus includes H1N1.

3. Use according to claim 1, characterized in that, The anti-influenza virus drug is used to inhibit influenza virus replication and internalization.

4. An anti-influenza virus drug composition, which is composed of 4-hydroxyderridin or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers and / or excipients.

5. The pharmaceutical composition of claim 4, wherein, The drug composition further contains one or more other anti-viral drugs.

6. The pharmaceutical composition of claim 5, wherein, The other anti-viral drugs are compounds and / or traditional Chinese medicine extracts with anti-viral activity, and / or traditional Chinese medicine compositions with anti-viral effect.

7. The pharmaceutical composition of claim 4, wherein, The drug composition is administered orally or by injection.

8. The pharmaceutical composition of claim 4, wherein, The preparation of the drug composition includes tablets, capsules, granules, dispersions, oral liquids, injections, sprays, and drops.

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