Application of chlorinated apidine in preparation of medicine for preventing and treating respiratory syncytial virus infection

Abstract

The invention belongs to the technical field of application of natural compounds, and relates to application of chlorinated apidine (APN) in preparation of a medicine for preventing and treating respiratory syncytial virus (RSV) infection. The invention also provides a pharmaceutical composition for resisting the respiratory syncytial virus. The pharmaceutical composition comprises an effective dose of chlorinated apidine. The invention discovers that APN has the anti-RSV activity effect for the first time. The APN can effectively inhibit RSV infection on Hep-2 and A549 cells, cytopathic effect tests and gene and protein expression detection find that the APN can effectively inhibit RSV proliferation, and an obvious dose-dependent effect is shown. In a reasonable dosage range, APN can be used as a natural and reliable antiviral compound for preparing a medicine for treating RSV infection or a medicine for preventing RSV infection.

Description

Technical Field

[0001] The invention belongs to the technical field of application of natural compounds, relates to a new application of apigenin chloride, and specifically relates to the application of apigenin chloride in the preparation of medicines. Background Art

[0002] Respiratory Syncytial Virus (RSV) is a common virus that mainly causes respiratory infections, especially posing a major health threat to infants, young children and the elderly. This virus mainly causes upper and lower respiratory tract infections, such as nasal congestion, cough, sore throat and other symptoms, but may also cause more serious diseases such as bronchiolitis and pneumonia.

[0003] RSV infection is very common worldwide. In addition to infants and young children, premature infants, individuals with compromised immune systems, and the elderly are also at high risk of developing serious complications after RSV infection.

[0004] RSV is spread through droplets, such as those released when coughing or sneezing, or by touching a surface contaminated with the virus and then touching a mucous membrane such as the mouth or nose. Symptoms of RSV infection usually appear within 4 to 6 days after exposure to the virus. Most people with RSV will have symptoms that resolve on their own within 1 to 2 weeks, but some high-risk groups may experience a longer course of the disease and require additional medical care.

[0005] There is currently no specific antiviral treatment to combat RSV infection. Researchers have conducted extensive research on RSV vaccine development, hoping to effectively prevent infection with the virus in the future. However, a vaccine with satisfactory results has not yet been developed. It is urgent to develop highly effective, non-toxic antiviral drugs based on natural compounds.

[0006] Apigeninidin Chloride (APN) is a naturally occurring flavonoid compound, a type of flavonol, mainly found in certain plants, such as the bran and leaf sheaths of red sorghum (Panicum miliaceum). This substance has attracted the attention of researchers for its unique biological activity and potential health benefits.

[0007] Red sorghum is an important food crop in my country. It can be used not only as food, but also as animal feed and raw material for brewing. In red sorghum, APN is one of its main pigment components, which gives red sorghum its unique color. In addition to its nutritional value, APN has been shown to have a wide range of biological activities. Studies have shown that APN has been reported to have significant antifungal and antioxidant activities, and can scavenge free radicals and protect cells from oxidative stress damage. In addition, it also has anti-inflammatory, antibacterial and potential anti-cancer effects. However, no relevant studies have reported on the antiviral effect of APN.

[0008] Currently, the treatment of RSV infection mainly relies on the antiviral drug ribavirin, but this drug has obvious toxic side effects such as loss of appetite, nausea, vomiting, diarrhea, etc., and there is no reliable vaccine for prevention and treatment. The emergence of clinical variant strains also makes the treatment of RSV infection face great challenges. So far, no commercial drug has been developed to resist RSV infection, so it is necessary to find a drug that effectively resists RSV infection to overcome the shortcomings in current practical applications. Summary of the invention

[0009] The purpose of the present invention is to provide a new pharmaceutical use of apigenin chloride, specifically the use of apigenin chloride in preparing a medicine for preventing and treating respiratory syncytial virus infection.

[0010] The invention also provides a pharmaceutical composition for resisting respiratory syncytial virus, which comprises an effective dose of apigenin chloride.

[0011] According to a further feature of the pharmaceutical composition of the present invention, the pharmaceutical composition also contains pharmaceutically acceptable excipients.

[0012] The present invention discovered for the first time that APN has anti-RSV activity. The research of the present invention shows that apigenin chloride (APN) can effectively inhibit RSV infection on Hep-2 and A549 cells, and through cytopathic test and gene and protein expression detection, it is found that APN can effectively inhibit the proliferation of RSV, showing a significant dose-dependent effect. Apigenin chloride has a significant inhibitory effect on the viral replication of RSV-infected Hep-2 cells, and has a significant inhibitory effect on the viral nucleic acid level, viral protein expression and cytopathic effect of the virus on Hep-2 cells, and the compound has no significant toxicity to Hep-2 cells in the concentration range of 0-200 μM. Therefore, within a reasonable dosage range, APN can be used as a natural and reliable antiviral compound for the preparation of drugs for the treatment or prevention of RSV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The toxicity of different concentrations of APN on Hep-2 and A549 cells is shown.

[0014] Figure 2 The effects of different concentrations of APN on RSV infection-induced pathological changes in Hep-2 and A549 cells are shown.

[0015] FIG. 3A to FIG. 3C Shown are the effects of different concentrations of APN on viral gene expression in RSV-infected Hep-2 and A549 cells.

[0016] FIG. 4A to FIG. 4C Shown are the effects of different concentrations of APN on viral protein expression in RSV-infected Hep-2 and A549 cells. DETAILED DESCRIPTION

[0017] In order to more clearly illustrate the present invention, the present invention is specifically described below in conjunction with specific embodiments. It is necessary to point out that this embodiment is only used to further illustrate the present invention. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0018] 1. Test Materials 1. Cells, strains, and compounds Human laryngeal cancer epithelial cells (Hep-2) and human lung cancer cells (A549) were cryopreserved by the applicant's laboratory, and the RSV A2 strain was provided by the pathogenic microbiology laboratory team of Jinan University. APN was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and the purity of the drug was ≥97%.

[0019] Hep-2 is a laryngeal cancer cell line derived from laryngeal cancer tissue, while A549 is a human lung adenocarcinoma cell line derived from lung cancer tissue. Since many viruses infect the human body through the respiratory tract, such as syncytial virus, influenza virus, coronavirus, etc., Hep-2 and A549 cells are often used as models to study these viruses.

[0020] 2. Main Reagents DMEM high-glucose medium was purchased from Hyclone; 0.25% trypsin-EDTA digestion solution (2.5 g porcine trypsin and 0.2 g EDTA dissolved in 1 L PBS, containing phenol red) was purchased from Solarbio; penicillin-streptomycin double antibody and trypsin were purchased from Solarbio; dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich, a subsidiary of Merck; CCK-8 kit was purchased from Beyotime, Shanghai; PBS (phosphate buffered saline) (NaCl 8.0 g; KCl 0.2 g; Na2HPO4 1.44 g; KH2PO4 0.24 g; distilled water to 1 L; pH adjusted to 7.4) used in the experiment was prepared by the laboratory; fetal bovine serum (FBS) was purchased from Gibio.

[0021] 3. Main instruments The biological safety cabinet, -80℃ ultra-low temperature refrigerator, multifunctional microplate reader and inverted microscope were all purchased from Thermo Fisher Scientific, USA; the fully automatic chemiluminescence image analysis system was purchased from Shanghai Tianneng Biotechnology Co., Ltd.; the electric constant temperature water bath was purchased from Changzhou Aohua Instrument Co., Ltd.; the cell culture incubator was purchased from Shanghai Precision Instrument Company; the low-speed automatic balancing centrifuge was purchased from Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.; EP tubes, cell culture bottles, cell culture plates and cryopreservation tubes were purchased from NEST, and the cell cryopreservation boxes were purchased from Sigma-Aldrich.

[0022] 2. Test methods 1. Cell Recovery 1) Add 10% fetal bovine serum (FBS) and 1% double antibody to the serum-free and double antibody-free DMEM high-glucose medium to obtain a complete medium for cell culture. 2) Take out the frozen Hep-2 cells and A549 cells from the liquid nitrogen tank and quickly place them in a 37°C constant temperature water bath to thaw.

[0023] 3) In the biosafety cabinet, transfer the cells to a new sterile centrifuge tube and add 3 mL of fresh culture medium to mix well. Set the centrifuge speed to 1000 rpm / min and centrifuge the cells for 5 min.

[0024] 4) After centrifugation, pour off the supernatant, add 2 mL of complete medium to the tube, gently pipette to resuspend the cell pellet, and then transfer the cells to a sterile 10 cm culture dish pre-added with 5 mL of complete medium.

[0025] 5) Gently shake the culture dish crosswise to evenly distribute the cells, place the cells in a 37°C constant temperature incubator containing 5% CO2, wait until the cells are completely attached to the wall, wash away the dead cells that are not attached to the wall, and then replace the culture dish with a fresh one to continue culturing the cells.

[0026] 2. Cell Passaging 1) When the cell growth density reaches above 80%-90%, remove the old culture medium in the culture dish and add PBS to wash the cells 1-2 times to remove the residual culture medium.

[0027] 2) Add 1 mL of pre-warmed 0.25% trypsin-EDTA digestion solution to the cell culture dish and observe the cells digested under a microscope until the outline becomes round. Then, remove the EDTA digestion solution and add 2 mL of complete culture medium to terminate the digestion process.

[0028] 3) Use a 1 mL pipette to suck up the culture medium and repeatedly blow all the cells off the bottom of the dish and transfer them to a 10 mL centrifuge tube. Set the centrifuge speed to 1000 rpm / min and centrifuge the cells for 5 min.

[0029] 4) After centrifugation, discard the supernatant and keep the cell pellet. Add 2 mL of complete medium and gently blow to mix the cell pellet. Then, subculture according to the experimental plan and add fresh complete medium to make the final volume reach 7 mL.

[0030] 3. Cell plating 1) When the cell density reaches about 80-90%, perform cell digestion as before.

[0031] 2) Determine the cell density and viability using a hemacytometer or automated cell counter to determine the number of cells required for inoculation.

[0032] 3) Press 4×10 5 Inoculate cells into each well of a six-well plate and gently shake the plate to evenly distribute the cells on the bottom of the plate. The amount of inoculum used depends on the desired cell density and the size of the plate.

[0033] 4) Place the plate in a 37°C incubator with 5% CO2 and culture overnight. When the cells are completely attached, proceed to the next step.

[0034] 4. Cytotoxicity Assay 1) Inoculate the cell suspension (about 1×10 4 Each well was filled with 100 μL of the solution, and different concentration groups ranging from 0 to 200 μM were designed, with 6 replicate wells in each concentration group. A blank group and a control group were also set up.

[0035] 2) Place the culture plate in a 37°C constant temperature incubator containing 5% CO2 for pre-culture for about 24 hours.

[0036] 3) Add different concentrations of APN (DMSO was added to the control group) to each well of the 96-well culture plate and incubate in an incubator for 72 h.

[0037] 4) Add 100 μL of serum-free culture medium containing 10% CCK-8 solution to each well. Try not to generate bubbles during the addition process to avoid affecting the OD value reading.

[0038] 5) Place the 96-well culture plate in the incubator and incubate for 1-2 h.

[0039] 6) Measure the absorbance (OD) at 450 nm using an enzyme-labeled instrument.

[0040] 7) Calculate cytotoxicity using the following formula: Cell viability (%) = [A (control) - A (experimental)] / [A (control) - A (blank)] × 100% Control: OD value of wells containing cells, culture medium, and CCK-8 Experiment: OD values ​​of wells containing cells, culture medium, CCK-8 and drug solutions Blank: OD value of wells containing culture medium and CCK-8 5. Virus Amplification 1) First, select Hep-2 cells with good growth conditions and inoculate them into T25 cell culture flasks. Continue to culture the cells to a density of about 70%. Add PBS and wash the cells three times to remove the residual complete culture medium. 2) Then, the RSV A2 strain at MOI = 0.01 was inoculated into Hep-2 cells, and the cells were placed in a cell culture incubator at 37°C and 5% CO2 to infect the cells for 2 h. The culture medium was replaced with fresh medium containing 2% FBS and continued to be cultured.

[0041] 3) When 80%-90% of cells become diseased, collect the virus culture medium and scrape the cell pellet into a 15 mL centrifuge tube.

[0042] 4) Repeat freezing and thawing in liquid nitrogen and constant temperature water bath for 3 times to release the virus particles in the cells. Set the centrifuge speed to 4000 rpm / min and centrifuge the cells for 10 min. Then mix the supernatant containing the virus and dispense it into 1.5mL EP tubes and store at -80℃ for subsequent experiments.

[0043] 6. APN antiviral test Preparation of drug diluent: APN drug powder was diluted to 200 mM with DMSO, and then further diluted with complete culture medium to obtain complete culture medium containing different concentrations of APN according to experimental requirements.

[0044] 1) First, select Hep-2 and A549 cells with good growth conditions and culture them at a rate of 2×10 5 Cells were seeded into each well of a 12-well plate.

[0045] 2) After the cells are completely attached, add RSV at an MOI of 0.1 and adsorb for 2 h in an incubator at 37°C and 5% CO2.

[0046] 3) The prepared complete culture medium containing 0 μM to 100 μM APN was added to the 12-well plates obtained in step (1) as different concentrations of APN groups. The 12-well plates were placed in a 37°C constant temperature cell culture incubator containing 5% CO2 and continued to be cultured for 72 hours.

[0047] 4) When the cells show obvious pathological changes, add nuclear staining solution to stain the nuclei, and use an inverted microscope to take pictures to record the cytopathic effect (CPE).

[0048] 6. APN inhibits RSV viral genes and proteins 1) Select Hep-2 and A549 cells with good growth conditions and culture them at 4×10 5 Cells were seeded into each well of a 6-well plate.

[0049] 2) After the cells are completely attached, add RSV at an MOI of 0.1 and adsorb for 2 h in an incubator at 37°C and 5% CO2, then replace with fresh culture medium and continue culturing.

[0050] 3) The prepared complete culture medium containing 0 μM to 100 μM APN was added to the 6-well plates obtained in step (1) as different concentrations of APN groups. The 6-well plates were placed in a 37°C constant temperature cell culture incubator containing 5% CO2 and continued to be cultured for 72 h.

[0051] 4) When the cells have obvious pathological changes, extract the total cell protein (or total RNA) and measure the protein concentration and RNA concentration for subsequent use.

[0052] 5) Perform Western blot or qPCR experiments to detect the expression of viral RSV-F and RSV-G proteins, as well as the expression of RSV-F, RSV-G and RSV-N viral genes.

[0053] 7. Data Analysis All experiments were repeated at least three times independently, and GraphPad Prism 9.0 software was used for data analysis and plotting, and the results were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used for statistical analysis. In the figure, * is used to indicate significant differences between groups, where * indicates P < 0.05, that is, significant differences; ** indicates P < 0.01, that is, very significant differences; *** indicates P < 0.001, that is, extremely significant differences.

[0054] 3. Results and Analysis 1. Determination of the maximum non-toxic concentration of APN No significant cytotoxicity was observed when the concentration of APN was 0-400 μM on Hep-2 cells, and no significant cytotoxicity was observed when the concentration of APN was 0-50 μM on A549 cells (e.g. Figure 1 The maximum non-toxic concentration of APN on Hep-2 cells is >400 μM, and the CC50 is greater than 400 μM. The maximum non-toxic concentration on A549 cells is about 50 μM, and the CC50 is 204.4 μM.

[0055] 2. Effect of APN on the pathological changes of RSV-infected Hep-2 and A549 cells like Figure 2 As shown, after treatment with 0 μM, 25 μM and 50 μM APN, the cytopathic effects of RSV-infected Hep-2 and A549 cells were significantly attenuated, and with the increase of APN concentration, the cytopathic effects were almost completely improved.

[0056] 3. Effect of APN on viral gene expression in RSV-infected Hep-2 cells like FIG. 3A to FIG. 3C As shown, with the gradual increase of APN concentration, the expression of RSV-F, RSV-G and RSV-N genes gradually decreased. Compared with the control group, the maximum decrease was approximately 80%, indicating that the ability of APN to inhibit RSV virus replication increased with the increase of its concentration, showing a dose-dependent effect.

[0057] 4. Effect of APN on viral protein expression in Hep-2 cells like FIG. 4A to FIG. 4C As shown, with the gradual increase of APN concentration, the expression of RSV-F and RSV-G viral proteins gradually decreased. Compared with the control group, the maximum reduction was approximately 90%, indicating that the ability of APN to inhibit RSV viral replication increased with the increase of its concentration, showing a dose-dependent effect.

[0058] IV. Pharmaceutical Compositions Containing APN APN has been reported as an anti-inflammatory, antibacterial and potential anti-cancer drug. The present invention first discovered that APN has anti-RSV activity.

[0059] The present invention also provides a pharmaceutical composition, which contains APN, and may also contain other active ingredients, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, and the pH value may vary depending on the properties of the formulated substances, and the technicians can adjust it as needed. The formulated pharmaceutical composition can be administered by conventional routes, including: intraperitoneal, intravenous or local administration.

[0060] The pharmaceutical composition of the present invention contains a safe and effective amount of APN and a pharmaceutically acceptable carrier. Such carriers include fillers, excipients, stabilizers, diluents, adhesives, lubricants, surfactants or combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0061] The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited thereto. It should be pointed out that for those skilled in the art and any technician familiar with the technical field, without departing from the overall concept of the present invention, the technical solution and the inventive concept of the present invention are equivalently replaced or changed, and several changes and improvements are made, which should also be regarded as the protection scope of the present invention.

Claims

1. Application of apigenin chloride in the preparation of medicines for preventing and treating respiratory syncytial virus infection.

2. A pharmaceutical composition for resisting respiratory syncytial virus, characterized in that: Includes an effective dose of apigenin chloride.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition also contains pharmaceutically acceptable excipients.

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