Application of astaxanthin in the preparation of antiviral products

Abstract

The present invention belongs to the technical field of antiviral drugs, and particularly relates to the application of astaxanthin in the preparation of antiviral products. The viruses include DNA viruses, positive single-stranded RNA viruses, and single-stranded negative-strand RNA viruses. Through in vitro and in vivo experiments, the present invention has confirmed that astaxanthin can inhibit virus adsorption and internalization, reduce the damage of the virus to the cell membrane structure and function, astaxanthin can inhibit virus invasion, and directly reduce the virus titer in animals. The research results of the present invention expand the scope of the efficacy of astaxanthin, and for the first time confirm that astaxanthin can prevent and treat virus-induced infections and diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of antiviral drugs, and particularly relates to the application of astaxanthin in the preparation of antiviral products. Background Art

[0002] Viruses are an important class of pathogens that cause human diseases. Viruses are the most important pathogens that cause human diseases. The World Health Organization (WHO) has announced that viral diseases have had a wide and profound impact on global public health and the economy.

[0003] Astaxanthin is a keto-carotenoid with the chemical name 3,3'-dihydroxy-4,4'-diketo-β,β'-carotene, abbreviated as AST. It is soluble in fats and organic solvents but insoluble in water. As a red pigment, it naturally exists in some plants and animals, making salmon and lobsters red and flamingo feathers pink. Most carotenoids represented by astaxanthin contain polyene chains and conjugated double bonds, which can quench singlet oxygen and scavenge ROS, thus terminating the chain oxidation reaction. The polymethylene olefin chain and end-ring structure of astaxanthin increase the rigidity of the cell membrane and at the same time change the cell membrane permeability. Its polar end can penetrate the cell membrane, thus increasing the strength and mechanical stability of the cell membrane. Therefore, it can limit the entry of peroxide-inducing factors into the cell, protect important molecules in the cell from oxidative damage, inhibit tumorigenesis, enhance the body's immunity, and also has a preventive effect on eye diseases caused by diabetes.

[0004] Currently, there is no research indicating that astaxanthin can resist viral infections. Summary of the Invention

[0005] Although certain progress has been made in the prevention and control of viral diseases, there is still a lack of corresponding prevention and control programs for some viral infections, which remains a major medical need at the present stage. The research results of the present invention show that the potential of astaxanthin as a novel broad-spectrum antiviral small molecule compound has emerged. Therefore, the purpose of the present invention is to broaden the scope of the efficacy of astaxanthin and provide its application in the preparation of antiviral products.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] The application of astaxanthin in the preparation of antiviral products, wherein the viruses include DNA viruses, positive-sense single-stranded RNA viruses, and single-stranded negative-sense RNA viruses. The DNA viruses include herpes simplex virus and hepatitis B virus. The positive-sense single-stranded RNA viruses include Zika virus, hand, foot, and mouth virus, and coronavirus. The single-stranded negative-sense RNA viruses include severe fever with thrombocytopenia syndrome virus and influenza A virus.

[0008] By measuring the transcription and expression levels of the virus in the human macrophage cell line THP-1, the results show that astaxanthin can inhibit the infection of severe fever with thrombocytopenia syndrome virus, Zika virus, coronavirus, influenza virus, hand, foot and mouth virus, hepatitis virus and herpes simplex virus in a time- and dose-dependent manner, and astaxanthin can reduce the accumulation of viral Np protein in THP-1 cells caused by the viral infection.

[0009] In some embodiments, the product is a drug.

[0010] In some embodiments, the astaxanthin is used to prepare a drug for preventing the viral infection.

[0011] In some embodiments, the astaxanthin is used to prepare a drug for treating the diseases caused by the viral infection.

[0012] The present invention proves through animal experiments that astaxanthin can reduce the mortality rate of mice caused by the viral infection.

[0013] In some embodiments, the astaxanthin is mixed with a carrier or excipient to prepare a pharmaceutically acceptable dosage form.

[0014] Furthermore, the drug is an oral preparation or an injection preparation.

[0015] In some embodiments, the oral preparation is a tablet, a capsule, a granule, a sustained-release preparation, or a dropping pill.

[0016] In some embodiments, the injection preparation is a solution type or a suspension type.

[0017] In some embodiments, the content of astaxanthin in the drug is 1 wt% - 99.9 wt%.

[0018] In some embodiments, the product is a health product. Currently, the astaxanthin health products on the market mainly include soft capsules, hard capsules, oral liquids and other forms. These products usually contain high-purity astaxanthin and are designed to provide antioxidant protection for consumers through oral administration. The health product provided by the present invention is designed to provide consumers with prevention of viral infection.

[0019] The beneficial effects of the present invention:

[0020] The present invention first discovers that astaxanthin can significantly inhibit the infection of severe fever with thrombocytopenia syndrome virus, Zika virus, coronavirus, influenza virus, hand, foot and mouth virus, hepatitis virus and herpes simplex virus. Further research shows that astaxanthin affects the mechanism of virus adsorption and internalization. The potential of astaxanthin as a new type of broad-spectrum antiviral small molecule compound has emerged, and animal experiments prove that astaxanthin can inhibit virus invasion and directly reduce the virus titer in animals. The results of the present invention provide new ideas for the development of broad-spectrum antiviral therapeutic drugs and nutritional health products for preventing virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 In:

[0022] Figure A shows the results of RT-qPCR detection of the virus titers in the extracellular and intracellular of THP-1 cells pretreated with different concentrations of AST for 4 hours and then treated with SFTSV at an MOI of 1 for 24 hours.

[0023] Figure B shows the results of RT-qPCR detection of the virus titers in the extracellular and intracellular of THP-1 cells pretreated with AST for 4 hours and then treated with SFTSV at an MOI of 1 for 12 and 24 hours.

[0024] Figure 2 In:

[0025] Figure A shows the results of RT-qPCR detection of the virus titers in the extracellular and intracellular of THP-1 cells pretreated with different concentrations of AST for 4 hours and then treated with HSV at an MOI of 1 for 24 hours.

[0026] Figure B shows the results of RT-qPCR detection of the virus titers in the extracellular and intracellular of THP-1 cells pretreated with AST for 4 hours and then treated with HSV at an MOI of 1 for 12 and 24 hours.

[0027] Figure 3 In:

[0028] Figure A shows the results of RT-qPCR detection of the virus titers in the extracellular and intracellular of THP-1 cells pretreated with different concentrations of AST for 4 hours and then treated with ZIKA at an MOI of 1 for 24 hours.

[0029] Figure B shows the results of RT-qPCR detection of the virus titers in the extracellular and intracellular of THP-1 cells pretreated with AST for 4 hours and then treated with ZIKA at an MOI of 1 for 12 and 24 hours.

[0030] Figure 4 In:

[0031] Figure of the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were pretreated with different concentrations of AST for 4 hours and then treated with HcoV at an MOI of 1 for 24 hours.

[0032] Figure of the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were pretreated with AST for 4 hours and then treated with HcoV at an MOI of 1 for 12 and 24 hours.

[0033] Figure 5 Among them:

[0034] Figure of the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were pretreated with different concentrations of AST for 4 hours and then treated with H1N1 at an MOI of 1 for 24 hours.

[0035] Figure of the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were pretreated with AST for 4 hours and then treated with H1N1 at an MOI of 1 for 12 and 24 hours.

[0036] Figure 6 Among them:

[0037] Figure of the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were pretreated with different concentrations of AST for 4 hours and then treated with EV71 at an MOI of 1 for 24 hours.

[0038] Figure of the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were pretreated with AST for 4 hours and then treated with EV71 at an MOI of 1 for 12 and 24 hours.

[0039] Figure 7 Among them:

[0040] Figure of the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were pretreated with different concentrations of AST for 4 hours and then treated with HBV at an MOI of 1 for 24 hours.

[0041] Figure of the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were pretreated with AST for 4 hours and then treated with HBV at an MOI of 1 for 12, 24, and 36 hours.

[0042] Figure 8 Among them:

[0043] Figure of the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were co-treated with different concentrations of AST and SFTSV at an MOI of 1 for 24 hours.

[0044] Panel B shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells treated with AST and SFTSV at an MOI of 1 for 12, 24, and 36 hours.

[0045] Figure 9 In:

[0046] Panel A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells co-treated with different concentrations of AST and HSV at an MOI of 1 for 24 hours.

[0047] Panel B shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells treated with AST and HSV at an MOI of 1 for 12, 24, and 36 hours.

[0048] Figure 10 In:

[0049] Panel A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells co-treated with different concentrations of AST and ZIKA at an MOI of 1 for 24 hours.

[0050] Panel B shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells treated with AST and ZIKA at an MOI of 1 for 12, 24, and 36 hours.

[0051] Figure 11 In:

[0052] Panel A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells co-treated with different concentrations of AST and HcoV at an MOI of 1 for 24 hours.

[0053] Panel B shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells treated with AST and HcoV at an MOI of 1 for 12, 24, and 36 hours.

[0054] Figure 12 In:

[0055] Panel A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells co-treated with different concentrations of AST and H1N1 at an MOI of 1 for 24 hours.

[0056] Panel B shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells treated with AST and H1N1 at an MOI of 1 for 12 and 24 hours.

[0057] Figure 13 In:

[0058] Figure A shows the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were co-treated with different concentrations of AST and EV71 with an MOI of 1 for 24 hours.

[0059] Figure B shows the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were treated with AST and EV71 with an MOI of 1 for 12 and 24 hours.

[0060] Figure 14 In:

[0061] Figure A shows the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were co-treated with different concentrations of AST and HBV with an MOI of 1 for 24 hours.

[0062] Figure B shows the results of RT-qPCR detection of extracellular and intracellular virus titers after THP-1 cells were treated with AST and HBV with an MOI of 1 for 12 and 24 hours.

[0063] Figure 15 In:

[0064] Figure A shows the protein expression level of SFTSV NP detected by Western blot after THP-1 cells were co-treated with SFTSV with an MOI of 1 and different concentrations of AST for 24 hours.

[0065] Figure B shows the protein expression level of SFTSV NP detected by Western blot after THP-1 cells were co-treated with SFTSV with an MOI of 1 and 10 μM AST for 12, 24, and 48 hours.

[0066] Figure C shows the infection level determined by the relative intensity of immunofluorescence images.

[0067] Figure D shows the quantitative statistics of the fluorescence intensity of the NP protein.

[0068] Figure 16 In:

[0069] Figure A shows the release of LDH in THP-1 cells after treatment with SFTSV and AST for 24 hours.

[0070] Figure B shows the cell Ca 2+ flux.

[0071] Figure C shows the cell membrane potential after treatment of THP-1 cells with SFTSV and AST.

[0072] Figure 17 In:

[0073] A is to calculate the r value through the TMA-DPH probe to reflect the effect of SFTSV on cell membrane fluidity at different times.

[0074] B is the change in membrane fluidity when cells are treated with SFTSV and 10 μM AST for 1 h.

[0075] Figure 18 In:

[0076] A is the expression level of Clathrin-H detected by Western blot after THP-1 cells are treated with SFTSV at MOI = 1 for 0.5 h, 1 h, and 2 h.

[0077] B is the inhibitory effect of AST on the expression of Clathrin-H when the expression level of Clathrin-H is the highest.

[0078] Figure 19 In:

[0079] A is the relative expression level of clathrin after THP-1 cells are treated with SFTSV and AST for 1 h.

[0080] B is the expression level of clathrin detected by Western blot.

[0081] Figure 20 is the expression level of all cell surface receptors detected by Western blot after THP-1 cells are treated with SFTSV and AST for 1 h.

[0082] Figure 21 In:

[0083] A is the phalloidin staining map of the cytoskeleton after THP-1 cells are treated with SFTSV and AST for 1 h.

[0084] B is the quantitative map of the actin expression level.

[0085] Figure 22 is the Kaplan-Meier survival curve of A129 mice infected with SFTSV.

[0086] Figure 23 is the expression level of SFTSV NP protein in tissues detected by Western blot. Specific embodiments

[0087] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0088] Example 1: Detection of the inhibitory effect of astaxanthin on virus infectivity.

[0089] I. Experimental materials:

[0090] Severe fever with thrombocytopenia syndrome bunyavirus, SFTSV, was kindly provided by the Jiangsu Provincial Center for Disease Control and Prevention. See https: / / doi.org / 10.1080 / 15548627.2024.2356505.

[0091] Zika virus, ZIKA, was kindly provided by the Jiangsu Provincial Center for Disease Control and Prevention.

[0092] Enterovirus 71, EV71, was kindly provided by the Jiangsu Provincial Center for Disease Control and Prevention.

[0093] Herpes simplex virus, HSV, was kindly provided by Professor Zhong Bo of Wuhan University.

[0094] Human coronavirus, HcoV, was kindly provided by Professor Zhang Leike of the Wuhan Institute of Virology.

[0095] Influenza A virus, H1N1, was kindly provided by Professor Zhang Leike of the Wuhan Institute of Virology.

[0096] Hepatitis B virus, HBV, was kindly provided by Professor Xia Yuchen of Wuhan University.

[0097] Dulbecco's Modified Eagle Medium: DMEM; Thermo Fisher, 11,965,092.

[0098] 1×SDS loading buffer: AbMloe BioScience, M5293.

[0099] Polyvinylidene fluoride membrane: Amersham, 10600023.

[0100] II. Experimental methods:

[0101] 1. Cell culture:

[0102] THP-1 cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C under 5% CO2. Virus-infected cells and virus-infected cells co-treated with AST were cultured in DMEM containing 2% fetal bovine serum and penicillin-streptomycin.

[0103] 2. Western blot:

[0104] Collect the cells treated with AST and virus infection, and lyse them with 1×SDS loading buffer supplemented with protease inhibitors. The extracted proteins are separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to a polyvinylidene difluoride membrane with a pore size of 0.45 μm. Wash the membrane with TBST washing buffer containing 5% non-fat milk powder at room temperature for 1 hour, and then incubate it overnight at 4°C with appropriately diluted primary antibody. Rinse the PVDF membrane three times with TBST washing buffer for 5 minutes each time, and then incubate it with HRP-conjugated secondary antibody at room temperature for 1 hour with a dilution ratio of 1:10000. Monitor the autoradiography signal on the ChemiDoc touch imaging system. β-actin is used as an internal control in the experiment.

[0105] 3. Confocal microscopy detection:

[0106] The treated cells are fixed with 4% methanol at room temperature for 10 minutes. After fixation, the cell membrane is permeabilized with a permeabilization solution containing 0.1% Triton X-100, and 1 mL of the permeabilization solution is added to each sample for 10 minutes. After permeabilization, block the non-specific binding of antibodies by incubating with 1 mL of PBS containing 2% BSA at room temperature for 30 min. Remove the blocking solution, add 200 μL of primary antibody diluted in PBS, and then incubate the samples containing the primary antibody in a chromatography cabinet at 4°C for 12 - 16 h. After permeabilization, add 1 mL of PBS containing 2% BSA to the samples and place them at room temperature for 30 minutes to block the non-specific binding of antibodies. Remove the blocking solution, add 200 μL of primary antibody diluted in PBS, and then incubate the samples containing the primary antibody in a chromatography cabinet at 4°C for 12 - 16 hours. After the incubation of the primary antibody is completed, remove the primary antibody, add 1 mL of pre-cooled PBS to wash the samples 3 times for 5 minutes each time. Dilute the fluorescently conjugated secondary antibody in PBS according to the dilution ratio, add 200 μL of the secondary antibody to each sample, and incubate it in the dark at room temperature for 30 minutes. Add 200 μL of nuclear stain DAPI and incubate it in the dark at room temperature for 5 minutes. Then pour out the DAPI staining solution and take pictures.

[0107] 4. RNA extraction and real-time fluorescence quantitative PCR:

[0108] Every 2×10 6Add one cell to 1 mL of TRIzol, then use a pipette tip to blow the cells to complete lysis, and transfer the cell lysate containing TRIzol to a 1.5 mL EP tube. Add 0.1 mL of chloroform substitute, cover the lid of the EP tube, and mix well on a vortex oscillator at room temperature for 3 minutes. Centrifuge at 10,000 g for 15 minutes at 4°C. After centrifugation, carefully take out the EP tube, and the RNA mainly remains in the upper aqueous phase. Carefully transfer the upper aqueous phase to a new centrifuge tube with a 200 μL pipette tip. Take about 500 μL of the upper aqueous phase, and do not transfer the middle layer. After transferring the upper aqueous phase, add 500 μL of isopropanol, invert and mix well, and let it stand at room temperature for 5 minutes. Then centrifuge the sample at 10,000 g for 10 minutes to remove the supernatant. Add 1 mL of 75% ethanol made from DEPC water to each tube, mix gently, cover the tube lid, centrifuge at 7500 g for 5 minutes, pour out the ethanol, air-dry the precipitate, and then add 50 μL of DEPC water to dissolve the RNA. Remove genomic DNA and reverse transcribe RNA according to the HiScript III RT SuperMix for qPCR kit. Establish a qPCR reaction system in a 96-well plate of an ABI fluorescence quantitative PCR instrument, and determine the copy number of the target gene in the test specimen by real-time detecting the fluorescence signal intensity changing with amplification, and obtain the Ct value.

[0109] III. Test Results:

[0110] To determine whether astaxanthin can inhibit virus infection, we selected several acute infectious RNA or DNA viruses respectively and measured the transcription and expression levels of the viruses in the human macrophage cell line THP-1. To more carefully determine the infection dose-dependent antiviral activity of AST, cells were infected with a virus at an MOI of 1. The results are as Figures 1 to 14 shown. We found that AST can inhibit the infection of severe fever with thrombocytopenia syndrome virus, Zika virus, coronavirus, influenza virus, hand, foot and mouth virus, hepatitis virus and herpes simplex virus in a time- and dose-dependent manner. In addition, confocal microscopy detection showed that after adding AST, the accumulation of viral Np protein in THP-1 cells caused by SFTSV infection was significantly reduced, as Figure 15 shown. Generally speaking, these results indicate that AST can be used as a broad-spectrum antiviral small molecule active substance.

[0111] Example 2: Astaxanthin alleviates the damage of virus to the cell membrane structure and function.

[0112] I. Test Methods:

[0113] 1. Plasma membrane potential measurement:

[0114] THP-1 cells were seeded into black / clear-bottom fluorescence 96-well plates and pre-incubated with 10 μM DiBAC4(3) dye for 30 minutes at 37 °C in the dark during the logarithmic growth phase, and then treated with AST and SFTSV for 30 minutes. After treatment, the waste liquid was discarded, and the cells were washed 3 times with PBS. Finally, 100 μL of PBS was added to each well, and the fluorescence value was read using a Spectra Max i3x microplate reader with an excitation wavelength of 493 nm and an emission wavelength of 516 nm.

[0115] 2. Detection of plasma membrane integrity:

[0116] The release amount of lactate dehydrogenase (LDH) was used to evaluate the plasma membrane integrity. Cells were seeded into six-well plates and cultured adherently for 24 hours, and then treated with SFTSV and astaxanthin for 24 hours. The supernatant and cells were collected separately; the supernatant was centrifuged at 250 g for 3 min at 4 °C, 20 μL was taken and pipetted into a transparent 96-well plate, and LDH detection solution was added respectively. After the reaction ended, the absorbance value at 450 nm was read on a SpectraMax i3x microplate reader; the total protein concentration of the cell pellet was detected using a BCA kit first, and the average LDH value was calculated according to the total LDH value of each sample. The total protein concentration of the precipitate was detected using a BCA kit first, the average LDH value was calculated according to the total LDH value of each sample, and the untreated group was used as t for normalization.

[0117] 3. Calcium ion Ca 2+ flux measurement:

[0118] The Fluo-3 / AM calcium ion fluorescence probe is a membrane-permeable probe that will be cleaved into Fluo-3 by intracellular esterase after entering the cytoplasm. Fluo-3 cannot cross the cell membrane and will therefore remain inside the cell. Cells were evenly seeded into fluorescence 96-well plates, adhered and exposed to astaxanthin and SFTSV for 24 hours, and then incubated with 5 μmol of Fluo-3 / AM probe for 30 minutes at 37 °C under weak light conditions; after loading the probe, the cells were washed 3 times with HBSS buffer, and finally 100 μL of HBSS buffer was added to each well, and then cultured in an incubator at 37 °C for 30 minutes to ensure that Fluo-3 / AM inside the cells was completely converted into Fluo-3.

[0119] 4. Plasma membrane fluidity test:

[0120] The membrane fluidity was measured using the TMA-DPH probe. TMA-DPH is cylindrical and sensitive to the repositioning caused by lipid interactions on the membrane, and is used for fluorescence polarization analysis in rotational motion studies. The fluorescence intensity of TMA-DPH itself is negative, and the fluorescence increases after binding to the membrane. The fluorescence anisotropy r is inversely proportional to the membrane mobility and is calculated by detecting the polarized fluorescence intensity of TMA-DPH. The specific experimental steps are as follows:

[0121] The cells were seeded in 100 mm culture dishes, allowed to adhere, and treated with SFTSV and AST for 4 h; the cells were incubated with 5 μM of the TMA-DPH probe in the dark at 37 °C for 20 min; the cells were collected, washed with HBSS buffer for 30 min, and then centrifuged for 3 min while keeping the cells as moist as possible; before detection, the cells were individually transferred onto slides and detected using a laser Raman spectrometer with a polarizer. Before detection, the cells were transferred onto slides and detected using a laser Raman spectrometer with a polarizer. The excitation wavelength was 325 nm, the emission wavelength was 430 nm, and the fluorescence values of the polarizer were calculated in the horizontal and vertical directions respectively.

[0122] 5. Detection of cytoskeletal integrity:

[0123] The cells were labeled with phalloidin probes for F-actin microfilament proteins to observe the cytoskeleton and cell morphology. The cells were seeded onto confocal culture dishes, allowed to adhere, treated with SFTSV and AST for 2 h, rinsed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and rinsed three times with PBS for 5 min each time to ensure no residual fixative; the cells were co-incubated with DAPI and phalloidin at room temperature for 15 min and rinsed three times with PBS for 5 min each time; the confocal culture dishes were placed under a laser confocal microscope for observation and photography. The confocal culture dishes were placed under a laser confocal microscope for observation and photography.

[0124] II. Test results:

[0125] To study the antagonistic effect of AST on membrane damage caused by virus internalization, we microscopically detected the plasma membrane potential, Ca 2+ flux, LDH release, plasma membrane fluidity, clathrin expression, and cytoskeletal network structure in cell experiments.

[0126] When the cells were exposed to the virus, the LDH release changed compared with untreated cells with or without astaxanthin added. Co-treatment with astaxanthin significantly alleviated the increase in LDH release caused by the virus, as Figure 16 shown in A, indicating that astaxanthin has a protective effect on membrane integrity.

[0127] Depolarization of the plasma membrane will further lead to changes in membrane permeability and extracellular Ca 2+ entry into the cells. Astaxanthin significantly inhibited the damage of the plasma membrane integrity by the virus. The Fluo-4 / AM probe is a fluorescent indicator of the permeability of intracellular calcium ions in cells, and the fluorescence intensity increases after binding to calcium ions. Astaxanthin restricted the calcium influx caused by the virus, as Figure 16 shown in B, indicating that astaxanthin has a protective effect on membrane stability.

[0128] The plasma membrane potential is a key indicator of the integrity and stability of the cell membrane. DiBAC4(3) is a slow-response probe sensitive to potential that can enter depolarized cells. Our detection results show that when cells are exposed to the virus, the membrane potential changes compared with untreated cells with or without astaxanthin added. Co-treatment with astaxanthin can significantly alleviate the abnormal membrane potential caused by the virus, and the results are as Figure 16 shown in C of

[0129] Changes in plasma membrane stability directly affect membrane fluidity. We used the hydrophobic fluorescent membrane probe TMA-DPH to detect membrane fluidity. TMA-DPH can anchor on the cell surface and localize in different regions of the phospholipid bilayer. By analyzing the fluorescence polarization values of TMA-DPH in the plasma membrane and membrane substructures, the fluidity of the cell membrane can be determined. As Figure 17 shown in A of Figure 17 , AST alone has the ability to reduce membrane fluidity, and the r value increases from 0.25 ± 0.0082 to 0.34 ± 0.017 after AST exposure. Compared with untreated cells, exposure to the virus increases the fluidity of the cell membrane, and the r value decreases from 0.25 ± 0.0082 to 0.11 ± 0.0047. Co-treatment with AST can restore the r value to near the background level, and the results are as

[0130] shown in B of Figure 18 . These data indicate that AST can alleviate the increase in cell membrane fluidity, rigidity and stability caused by the virus, and ultimately change the endocytosis process of the virus. Figure 19 Previous studies have shown that many viruses enter cells through clathrin-mediated endocytosis. Therefore, we detected the expression level of clathrin to find the peak expression of clathrin during virus internalization. First, we treated cells with astaxanthin for 0.5 hours, 1 hour and 2 hours. From the results, it can be seen that clathrin is recruited to the maximum extent at 0.5 hours, and the results are as Figure 20 shown. Therefore, we harvested proteins 0.5 hours after adding the virus. Western blot results showed that the expression of clathrin increased significantly after adding the virus, and the addition of astaxanthin significantly inhibited the increase in clathrin expression, and the results are as

[0131] shown in Figure 21As shown in A of Figure 21 , in the control group and the group treated with AST alone, F-actin showed well-extended stress fibers with a certain thickness between the cell surface and the cytoplasm. After virus treatment, F-actin was disrupted and appeared disorganized. These actin filaments formed an anisotropic network and disordered bundles, aggregating in the area near the cell membrane and at the edges of lamellipodia and filopodia. After combined treatment with AST, the number and length of these protrusions were significantly reduced, and the highly polarized F-actin was also alleviated. We quantified the fluorescence intensity of actin, and the results are shown in

[0132] B of . The above results indicate that AST can reduce virus-induced F-actin assembly and phagocytosis.

[0133] Example 3: Astaxanthin inhibits virus infection in vivo.

[0134] I. Test method:

[0135] 1. Animal experiment:

[0136] 1000 PFU of SFTSV, HSV or ZIKA was diluted in 100 mL of DMEM and intraperitoneally injected into male and female mice aged 6 - 8 weeks. At the same time, mice were injected with DMEM as a control. The survival rate, body weight loss and disease symptoms of the mice were monitored daily to record the clinical course of virus infection. In the in vivo antiviral test, male and female mice aged 6 - 8 weeks were infected with 1000 PFU of SFTSV, HSV or ZIKA. The AST treatment group was treated with 150 mg / kg / day of AST daily, and the vehicle was used as a control. The vehicle composition was 5% DMSO + 5% ethanol + 40% PEG400 + 50% normal saline. For tissue localization and pathological analysis, all mice were anesthetized, imaged and tissue samples were collected at the designated time points.

[0137] II. Test results:

[0138] We investigated whether AST could prevent and control mice from being infected with SFTSV. Preliminary studies were conducted on A129 mice to determine whether AST pretreatment and co-treatment showed similar disease progression to the control group. A129 mice aged 6 to 8 weeks were infected with SFTSV at an MOI of 5 via the intraperitoneal route, and the weight loss and mortality of these mice and mock-infected mice were monitored. By day 6, two out of five mice in the SFTSV-treated group had died, and all died on the seventh day. Mice inoculated with the same dose of virus and given AST co-treatment did not show typical clinical symptoms of the disease, and 60% of this group survived. Mice inoculated with the same dose of virus and given AST pretreatment did not show typical clinical symptoms of the disease, and 80% of this group survived. The results are as Figure 22 shown, while the survival rate of the blank control and astaxanthin treatment groups was 100%. Then we performed immunohistochemistry and WB detection on the NP protein in the spleen tissue. The results showed that AST significantly inhibited the virus titer in mice. The results are as Figure 23 shown.

[0139] Observation of mice in all groups found that mice given SFTSV showed significant weight loss, while the body weight of mice given AST remained almost unchanged. However, HSV and Zika did not cause significant changes in the body weight of mice. In addition, H&E staining was used to evaluate the pathological changes induced by HSV, SFTSV, and Zika infections in organs. In mice infected with SFTSV, the overall structure of the spleen tissue was abnormal, the boundary between the white pulp and red pulp was unclear, the red pulp decreased sharply, the structure of the lymphoid nodules in the white pulp was unclear, many lymphocytes were visible, obvious necrosis was observed, obvious pyknosis and fragmentation of the cell nuclei were visible, and a small number of multinucleated giant cells were visible. The overall structure of the spleen tissue of mice treated with AST was slightly abnormal, the boundary between the red pulp and white pulp was distinct, the structure of the splenic nodules was clear, no obvious atrophy or shrinkage of the splenic nodules was observed, no obvious hyperplasia of connective tissue was observed in the tissue stroma, and a small number of multinucleated giant cell infiltrations were observed in the tissue. All these results indicate that AST can reduce the lethality and improve the outcome of virus infection in mice.

[0140] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concepts. Obviously, those skilled in the art can make various changes and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and modifications.

Claims

1. Use of astaxanthin as the only active ingredient in the preparation of antiviral products, characterized in that, The virus is severe fever with thrombocytopenia syndrome virus.

2. The application of astaxanthin as the sole active ingredient in the preparation of antiviral products according to claim 1, wherein The product is a drug.

3. The application of astaxanthin as the only active ingredient in the preparation of antiviral products according to claim 2, characterized in that, The astaxanthin is used for preparing a drug for preventing virus infection.

4. The application of astaxanthin as the only active ingredient in the preparation of antiviral products according to claim 2, wherein The astaxanthin is used for preparing a drug for treating diseases caused by virus infection.

5. The application of astaxanthin as the only active ingredient in the preparation of antiviral products according to claim 2, characterized in that, The astaxanthin and a carrier or excipient are mixed to prepare a pharmaceutically acceptable dosage form.

6. The application of astaxanthin as the only active ingredient in the preparation of antiviral products according to claim 5, characterized in that, The drug is an oral preparation or an injection preparation.

7. Use of astaxanthin as the only active ingredient in the preparation of an antiviral product according to claim 6, characterized in that, The oral preparation is a tablet, a capsule, a granule, a sustained-release preparation or a dropping pill.

8. The application of astaxanthin as the only active ingredient in the preparation of antiviral products according to claim 6, wherein, The injection preparation is a solution type or a suspension type.

9. The use of astaxanthin as the sole active ingredient in the preparation of an antiviral product according to claim 6, characterized in that, The content of astaxanthin in the drug is 1 wt% - 99.9 wt%.

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