Application of astaxanthin in preparation of anti-coronavirus product

Abstract

The invention belongs to the technical field of antiviral drugs, and particularly relates to application of astaxanthin in preparation of an anti-coronavirus product. In-vitro and in-vivo experiments prove that the astaxanthin can inhibit virus adsorption and internalization and relieve damage of viruses to cell membrane structures and functions, the astaxanthin can inhibit virus invasion and directly reduce virus titer in animal bodies, the research result of the invention expands the efficacy range of the astaxanthin, and the astaxanthin can be used for preparing the medicine for treating animal diseases. The astaxanthin is proved to be capable of preventing and treating infection and diseases caused by viruses for the first time.

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 anti-coronavirus products.

[0002] This invention is a divisional application of application number 202411682624.6, and the application date is November 22, 2024. Background Art

[0003] Viruses are a major class of pathogens that cause human disease. They are the most important pathogens causing human disease. The World Health Organization (WHO) has declared that viral diseases have a widespread and profound impact on global public health and the economy.

[0004] Astaxanthin, a ketocarotenoid with the chemical name 3,3′-dihydroxy-4,4′-diketo-β,β′-carotene (AST), is soluble in fats and organic solvents but insoluble in water. It occurs naturally in some plants and animals as a red pigment, giving salmon and lobster their red color and flamingo feathers their pink hue. Most carotenoids, such as astaxanthin, contain polyene chains and conjugated double bonds, which can quench singlet oxygen and scavenge ROS, thereby terminating chain oxidation reactions. Astaxanthin's polymethylene olefin chains and terminal ring structure increase cell membrane rigidity and permeability. Its polar end can penetrate the cell membrane, thereby increasing its strength and mechanical stability. Consequently, it can restrict the entry of peroxide-initiating factors into cells, protecting important cellular molecules from oxidative damage, inhibiting tumorigenesis, enhancing immunity, and preventing diabetic eye diseases.

[0005] There are currently no studies showing that astaxanthin can fight viral infections. Summary of the Invention

[0006] Despite progress in preventing and controlling viral diseases, there is still a lack of effective prevention and control options for some viral infections, which remains a major medical need. The present invention demonstrates the potential of astaxanthin as a novel broad-spectrum antiviral small molecule. Therefore, the present invention aims to broaden the efficacy of astaxanthin and provide its application in the preparation of antiviral products.

[0007] To achieve the above object, the present invention adopts the following technical solutions: Astaxanthin is used as the sole active ingredient in the preparation of antiviral products. The viruses include DNA viruses, positive-sense single-stranded RNA viruses and single-stranded negative-strand 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 disease virus and coronavirus. The single-stranded negative-strand RNA viruses include fever with thrombocytopenia syndrome virus and influenza A virus.

[0008] The present invention measured the transcription and expression levels of viruses in the human macrophage cell line THP-1, and the results showed that astaxanthin can inhibit the infection of 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 pharmaceutical.

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

[0011] In some embodiments, the astaxanthin is used to prepare a medicament for treating the disease 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 virus 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 injectable preparation.

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

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

[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 supplement. Currently, astaxanthin health supplements on the market mainly include soft capsules, hard capsules, and oral liquids. These products generally contain high-purity astaxanthin and are intended to provide consumers with antioxidant protection through oral administration. The health supplement provided by the present invention is intended to provide consumers with protection against viral infections.

[0019] Beneficial effects of the present invention: The present invention discovers for the first time that astaxanthin can significantly inhibit infections with fever with thrombocytopenia syndrome virus, Zika virus, coronavirus, influenza virus, hand, foot and mouth virus, hepatitis virus, and herpes simplex virus. Further studies have shown the mechanism by which astaxanthin affects viral adsorption and internalization. The potential of astaxanthin as a new broad-spectrum antiviral small molecule compound has been revealed, and animal experiments have shown that astaxanthin can inhibit viral invasion and directly reduce viral titers 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 viral infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 middle: A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells after they were pretreated with different concentrations of AST for 4 hours and then treated with SFTSV at an MOI of 1 for 24 hours.

[0021] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells after they were pretreated with AST for 4 hours and then treated with SFTSV at an MOI of 1 for 12 and 24 hours.

[0022] Figure 2 middle: A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells after they were pretreated with different concentrations of AST for 4 hours and then treated with HSV at an MOI of 1 for 24 hours.

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

[0024] Figure 3 middle: A shows the results of RT-qPCR detection of extracellular and intracellular viral titers after THP-1 cells were pretreated with different concentrations of AST for 4 hours and then treated with ZIKA at an MOI of 1 for 24 hours.

[0025] B shows the results of RT-qPCR detection of extracellular and intracellular viral titers after THP-1 cells were pretreated with AST for 4 hours and then treated with ZIKA at an MOI of 1 for 12 and 24 hours.

[0026] Figure 4 middle: A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells after they were pretreated with different concentrations of AST for 4 hours and then treated with HcoV at an MOI of 1 for 24 hours.

[0027] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells after they were pretreated with AST for 4 hours and then treated with HcoV at an MOI of 1 for 12 and 24 hours.

[0028] Figure 5 middle: A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells after they were pretreated with different concentrations of AST for 4 hours and then treated with H1N1 at an MOI of 1 for 24 hours.

[0029] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells after they were pretreated with AST for 4 hours and then treated with H1N1 at an MOI of 1 for 12 and 24 hours.

[0030] Figure 6 middle: A shows the results of RT-qPCR detection of extracellular and intracellular viral titers in THP-1 cells after they were pretreated with different concentrations of AST for 4 hours and then treated with EV71 at an MOI of 1 for 24 hours.

[0031] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells after they were pretreated with AST for 4 hours and then treated with EV71 at an MOI of 1 for 12 and 24 hours.

[0032] Figure 7 middle: A shows the results of RT-qPCR detection of extracellular and intracellular viral titers in THP-1 cells after they were pretreated with different concentrations of AST for 4 hours and then treated with HBV at an MOI of 1 for 24 hours.

[0033] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells after they were pretreated with AST for 4 hours and then treated with HBV at an MOI of 1 for 12, 24, and 36 hours.

[0034] Figure 8 middle: A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells treated with different concentrations of AST and SFTSV at an MOI of 1 for 24 hours.

[0035] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers of THP-1 cells treated with AST and SFTSV at an MOI of 1 for 12, 24, and 36 hours.

[0036] Figure 9 middle: A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells treated with different concentrations of AST and HSV at an MOI of 1 for 24 hours.

[0037] 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.

[0038] Figure 10 middle: A shows the results of RT-qPCR detection of extracellular and intracellular viral titers in THP-1 cells treated with different concentrations of AST and ZIKA at an MOI of 1 for 24 hours.

[0039] 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.

[0040] Figure 11 middle: A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells treated with different concentrations of AST and HcoV at an MOI of 1 for 24 hours.

[0041] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers of THP-1 cells treated with AST and HcoV at an MOI of 1 for 12, 24, and 36 hours.

[0042] Figure 12 middle: A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells treated with different concentrations of AST and H1N1 at an MOI of 1 for 24 hours.

[0043] 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.

[0044] Figure 13 middle: A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells treated with different concentrations of AST and EV71 at an MOI of 1 for 24 hours.

[0045] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells treated with AST and EV71 at an MOI of 1 for 12 and 24 hours.

[0046] Figure 14middle: A shows the results of RT-qPCR detection of extracellular and intracellular virus titers in THP-1 cells treated with different concentrations of AST and HBV at an MOI of 1 for 24 hours.

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

[0048] Figure 15 middle: A: THP-1 cells were co-treated with SFTSV at an MOI of 1 and different concentrations of AST for 24 h, and the protein expression of SFTSV NP was detected by Western blot.

[0049] B: THP-1 cells were co-treated with SFTSV at an MOI of 1 and 10 μM AST for 12, 24, and 48 hours, respectively, and the protein expression of SFTSV NP was detected by Western blot.

[0050] C is the infection level determined by the relative intensity of immunofluorescence images.

[0051] D is the quantitative statistics of NP protein fluorescence intensity.

[0052] Figure 16 middle: A shows the release of cellular LDH in THP-1 cells after treatment with SFTSV and AST for 24 h.

[0053] B is the cellular Ca of THP-1 cells after treatment with SFTSV and AST for 24 h. 2+ flux.

[0054] C is the cell membrane potential of THP-1 cells after treatment with SFTSV and AST.

[0055] Figure 17 middle: A is the r value calculated by TMA-DPH probe, reflecting the effect of SFTSV on cell membrane fluidity at different times.

[0056] B shows the changes in membrane fluidity when cells were treated with SFTSV and 10 μM AST for 1 h.

[0057] Figure 18 middle: A: The expression level of Clathrin-H was detected by Western blot after THP-1 cells were treated with SFTSV at MOI=1 for 0.5h, 1h and 2h.

[0058] B shows the inhibitory effect of AST on Clathrin-H expression when the Clathrin-H expression level is the highest.

[0059] Figure 19 middle: A is the relative expression level of clathrin in THP-1 cells after treatment with SFTSV and AST for 1 h.

[0060] B: Western blot detection of clathrin expression.

[0061] Figure 20 Western blot was used to detect the expression levels of all cell surface receptors in THP-1 cells after treatment with SFTSV and AST for 1 hour.

[0062] Figure 21 middle: A is the phalloidin staining of the cytoskeleton of THP-1 cells after treatment with SFTSV and AST for 1 h.

[0063] B is a quantitative graph of actin expression levels.

[0064] Figure 22 Figure 2 is the Kaplan-Meier survival curve of A129 mice infected with SFTSV.

[0065] Figure 23 Western blot was used to detect the expression level of SFTSV NP protein in tissues. DETAILED DESCRIPTION

[0066] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0067] Example 1: Detection of the inhibitory effect of astaxanthin on viral infectivity.

[0068] 1. Test materials: SFTSV was a gift from Jiangsu Provincial Center for Disease Control and Prevention, see https: / / doi.org / 10.1080 / 15548627.2024.2356505.

[0069] Zika virus, ZIKA, was a gift from Jiangsu Provincial Center for Disease Control and Prevention.

[0070] Hand, foot and mouth virus, EV71, was a gift from Jiangsu Provincial Center for Disease Control and Prevention.

[0071] Herpes simplex virus, HSV, was gifted by Professor Zhong Bo from Wuhan University.

[0072] Human coronavirus, HcoV, was gifted by Professor Zhang Lei Ke of Wuhan Institute of Virology.

[0073] Influenza A virus, H1N1 was donated by Professor Zhang Lei Ke from Wuhan Institute of Virology.

[0074] Hepatitis B virus (HBV) was donated by Professor Xia Yuchen from Wuhan University.

[0075] Dulbecco's modified Eagle's medium (DMEM); Thermo Fisher, 11,965,092.

[0076] 1× SDS wash buffer: AbMloe BioScience, M5293.

[0077] Polyvinylidene fluoride membrane: Amersham, 10600023.

[0078] 2. Test methods: 1. Cell culture: THP-1 cells were cultured in Dulbecco's modified medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 µg / mL streptomycin at 37°C in a humidified atmosphere of 5% CO2. Virus-infected cells and cells co-treated with virus infection and AST were cultured in DMEM supplemented with 2% fetal bovine serum and penicillin-streptomycin.

[0079] 2. Western blotting: Cells treated with AST and virus infection were harvested and lysed with 1× SDS-ionization buffer supplemented with protease inhibitors. The extracted proteins were separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to a 0.45 μm pore size polyvinylidene difluoride membrane. The membranes were washed with TBST-containing 5% nonfat dry milk for 1 hour at room temperature and then incubated with appropriately diluted primary antibodies overnight at 4°C. The PVDF membranes were rinsed three times with TBST-containing wash buffer for 5 minutes each and then incubated with HRP-conjugated secondary antibodies at a dilution of 1:10,000 for 1 hour at room temperature. Autoradiographic signals were monitored on a ChemiDoc touch imaging system. β-actin was used as an internal control.

[0080] 3. Confocal microscopy detection: Treated cells were fixed with 4% methanol for 10 minutes at room temperature. The fixed cell membranes were then incubated with 0.1% Triton X-100. 1 mL of permeabilization buffer was added to each sample for 10 minutes. After permeabilization, the samples were blocked with 1 mL of 2% BSA in PBS for 30 minutes at room temperature to block nonspecific antibody binding. The blocking buffer was removed, and 200 µL of primary antibody diluted in PBS was added. The samples containing primary antibody were then incubated in a chromatography cabinet at 4°C for 12–16 hours. After permeabilization, 1 mL of 2% BSA in PBS was added to the samples and incubated at room temperature for 30 minutes to block nonspecific antibody binding. The blocking buffer was removed, and 200 µL of primary antibody diluted in PBS was added. The samples containing primary antibody were then incubated in a chromatography cabinet at 4°C for 12–16 hours. After the primary antibody incubation was complete, the primary antibody was removed, and the samples were rinsed three times with 1 mL of pre-chilled PBS for 5 minutes each. Dilute the fluorescein-conjugated secondary antibody in PBS according to the dilution ratio. Add 200µL of secondary antibody to each sample and incubate at room temperature in the dark for 30 minutes. Add 200µL of the nuclear stain DAPI and incubate at room temperature in the dark for 5 minutes. Discard the DAPI staining solution and photograph.

[0081] 4. RNA extraction and real-time fluorescence quantitative PCR: Every 2×10 6 Add 1 mL of TRIzol to each cell. Complete cell lysis by blowing the cells with a pipette tip. Transfer the TRIzol-containing cell lysate to a 1.5 mL Eppendorf tube. Add 0.1 mL of chloroform substitute, cap the tube, and mix thoroughly on a vortex for 3 minutes at room temperature. Centrifuge at 10,000 g for 15 minutes at 4°C. After centrifugation, carefully remove the Eppendorf tube, leaving the RNA primarily in the upper aqueous phase. Using a 200 μL pipette tip, carefully transfer approximately 500 μL of the upper aqueous phase to a new centrifuge tube, avoiding the middle phase. After removing the upper aqueous phase, add 500 μL of isopropanol, invert to mix, and let stand at room temperature for 5 minutes. Then, centrifuge the sample at 10,000 g for 10 minutes and remove the supernatant. Add 1 mL of 75% ethanol prepared from DEPC water to each tube, mix gently, cap the tube, and centrifuge at 7500 g for 5 minutes. Discard the ethanol, air-dry the pellet, and then add 50 μL of DEPC water to dissolve the RNA. Genomic DNA removal and RNA reverse transcription are performed using the HiScript III RT SuperMix for qPCR Kit. A qPCR reaction system is established in a 96-well plate on an ABI fluorescent quantitative PCR instrument. The copy number of the target gene in the sample is determined by real-time monitoring of the fluorescence signal intensity that changes with amplification, and the Ct value is obtained.

[0082] 3. Test results: To determine whether astaxanthin can inhibit viral infection, we selected several acutely infectious RNA or DNA viruses 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 viruses at an MOI of 1. The results are shown in Figure 2. Figures 1 to 14 As shown in Figure 2, we found that AST could inhibit the infection of 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 showed that after the addition of AST, SFTSV infection led to a significant reduction in the accumulation of viral Np protein in THP-1 cells, as shown in Figure 2. Figure 15 Overall, these results indicate that AST can be used as a broad-spectrum antiviral small molecule active substance.

[0083] Example 2: Astaxanthin reduces viral damage to cell membrane structure and function.

[0084] 1. Test method: 1. Plasma membrane potential determination: THP-1 cells were seeded into black / clear bottom fluorescent 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. The cells were then treated with AST and SFTSV for 30 minutes. The cells were then washed three times with PBS, and 100 μL of PBS was added to each well. Fluorescence was read using a Spectra Max i3x microplate reader with an excitation wavelength of 493 nm and an emission wavelength of 516 nm.

[0085] 2. Plasma membrane integrity detection: LDH release was used to assess plasma membrane integrity. Cells were seeded into six-well plates and allowed to adhere for 24 hours before being treated with SFTSV and astaxanthin for 24 hours. The supernatant and cells were collected separately. The supernatant was centrifuged at 250g for 3 minutes at 4°C, and 20 μL of the supernatant was pipetted into a clear 96-well plate. LDH detection solution was then added. After the reaction, the absorbance at 450 nm was read on a SpectraMax i3x microplate reader. The total protein concentration of the cell pellet was first determined using a BCA assay kit, and the average LDH value was calculated based on the total LDH value of each sample. The average LDH value was normalized to the untreated group as t.

[0086] 3. Calcium ion Ca 2+ Flux determination: The Fluo-3 / AM calcium ion fluorescent probe is a membrane-permeable probe that is cleaved into Fluo-3 by intracellular esterases upon entering the cytoplasm. Fluo-3 cannot cross the cell membrane and therefore remains within the cell. Cells were evenly seeded into a fluorescent 96-well plate and exposed to astaxanthin and SFTSV for 24 hours. They were then incubated with 5 μmol of Fluo-3 / AM probe at 37°C under low light for 30 minutes. After probe loading, the cells were rinsed three times with HBSS buffer, and finally, 100 μL of HBSS buffer was added to each well, followed by incubation in a 37°C incubator for 30 minutes to ensure complete conversion of intracellular Fluo-3 / AM to Fluo-3.

[0087] 4. Plasma membrane fluidity test: Membrane fluidity is measured using the TMA-DPH probe. TMA-DPH is cylindrical and sensitive to reorientation caused by lipid interactions on the membrane, making it useful for fluorescence polarization analysis in rotational motion studies. TMA-DPH itself has a negative fluorescence intensity, but its fluorescence increases upon membrane binding. Fluorescence anisotropy, r, is inversely proportional to membrane mobility and is calculated by measuring the polarized fluorescence intensity of TMA-DPH. The specific experimental steps are as follows:

[0088] Cells were seeded in 100 mm culture dishes, allowed to adhere, and treated with SFTSV and AST for 4 hours. Cells were then incubated with 5 μM TMA-DPH probe at 37°C in the dark for 20 minutes. Cells were harvested, washed with HBSS buffer for 30 minutes, and then centrifuged for 3 minutes, keeping the cells moist as much as possible. Prior to detection, cells were individually transferred to glass slides and examined using laser Raman spectroscopy with a polarizer. Prior to detection, cells were transferred to glass slides and examined using a laser Raman spectrometer with a polarizer, using an excitation wavelength of 325 nm and an emission wavelength of 430 nm. Fluorescence values were calculated for the horizontal and vertical directions of the polarizer.

[0089] 5. Cytoskeleton integrity detection: Phalloidin was used to label cell F-actin microfilaments to observe the cytoskeleton and morphology. Cells were seeded onto confocal culture dishes, allowed to adhere, and treated with SFTSV and AST for 2 hours. The cells were then rinsed three times with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. The cells were then rinsed three times with PBS for 5 minutes each to ensure that no residual fixative remained. The cells were incubated with DAPI and phalloidin for 15 minutes at room temperature and rinsed three times with PBS for 5 minutes each. The confocal culture dishes were then placed under a laser confocal microscope for observation and photography.

[0090] 2. Test results: In order to study the antagonistic effect of AST on membrane damage caused by virus internalization, we detected the plasma membrane potential, Ca 2+ flux, LDH release, plasma membrane fluidity, clathrin expression, and cytoskeletal network structure.

[0091] When cells were exposed to viruses, LDH release was altered compared to untreated cells with or without astaxanthin. Co-treatment with astaxanthin significantly alleviated the virus-induced increase in LDH release, such as Figure 16 As shown in A, it shows that astaxanthin has a protective effect on membrane integrity.

[0092] Depolarization of the plasma membrane further leads to changes in membrane permeability and extracellular Ca 2+ Enter the cell. Astaxanthin significantly inhibited the virus's damage to the integrity of the plasma membrane. Fluo-4 / AM probe is a fluorescent indicator of cell permeability to intracellular calcium ions. The fluorescence intensity increases after binding to calcium ions. Astaxanthin limits the influx of calcium ions caused by viruses, such as Figure 16 As shown in B, it shows that astaxanthin has a protective effect on membrane stability.

[0093] Plasma membrane potential is a key indicator of cell membrane integrity and stability. DiBAC4 (3) is a slow-acting probe sensitive to potential that can enter depolarized cells. Our results showed that when cells were exposed to viruses, the membrane potential changed compared to untreated cells with or without astaxanthin. Co-treatment with astaxanthin significantly alleviated the membrane potential abnormalities caused by viruses, as shown in the following table. Figure 16 As shown in C.

[0094] 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 locate 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. Figure 17 As shown in Figure A, AST alone was able to reduce membrane fluidity, with the r value increasing from 0.25 ± 0.0082 to 0.34 ± 0.017 after AST exposure. Compared with untreated cells, exposure to the virus increased the fluidity of the cell membrane, with the r value decreasing from 0.25 ± 0.0082 to 0.11 ± 0.0047. The combined use of AST restored the r value to near background levels, as shown in Figure 4. Figure 17 These data suggest that AST can alleviate the virus-induced increase in cell membrane fluidity, decrease in rigidity and stability, and ultimately alter the viral endocytosis process.

[0095] Previous studies have shown that many viruses enter cells through clathrin-mediated endocytosis, so we detected the expression level of clathrin to find the peak expression of clathrin during virus internalization. First, we treated the cells with astaxanthin for 0.5 hours, 1 hour and 2 hours. From the results, we can see that clathrin is recruited to the maximum extent at 0.5 hours. The results are as follows Figure 18 Therefore, we harvested the protein 0.5 hours after adding the virus. Western blot results showed that the expression of clathrin increased significantly after adding the virus. The addition of astaxanthin significantly inhibited the increase in clathrin expression. Figure 19 Then we tested all membrane receptors of SFTSV, and the results showed that AST could inhibit the expression of all cell surface receptors induced by SFTSV. Figure 20 shown.

[0096] Microfilaments are commonly found in various plant tissue cells, and their polymerization and depolymerization processes are closely related to various life activities such as cell morphology maintenance, cell movement, cytoplasmic circulation, material transport, apical growth and signal transduction. Therefore, the staining of F-actin by phalloidin was used to evaluate the effect of cell absorption of viruses on the skeleton. Figure 21 As shown in Figure A, in the control group and the group treated with AST alone, F-actin showed a certain amount of thick bundles of well-stretched stress fibers between the cell surface and the cytoplasm. After virus treatment, F-actin was destroyed and appeared disorganized. These actins formed anisotropic networks and disorganized filaments, which gathered in areas close to the cell membrane and at the edges of the lamellipodia and filopodia. After combined treatment with AST, the number and length of these protrusions were significantly reduced, and the high polarization of F-actin was also alleviated. We quantified the fluorescence intensity of actin, and the results are shown in Figure 2. Figure 21 As shown in B. The above results indicate that AST can reduce virus-induced F-actin assembly and phagocytosis.

[0097] All the above results indicate that AST can directly act on the cell membrane, stabilize the phospholipid bilayer, reduce fluidity, limit the virus entry process and intracellular accumulation, and directly reduce the adverse effects of a large number of viruses on target cells and tissues.

[0098] Example 3: Astaxanthin inhibits viral infection in vivo.

[0099] 1. Test method: 1. Animal experiments: 1000 PFU of SFTSV, HSV, or ZIKA was diluted in 100 mL of DMEM and injected intraperitoneally into 6-8 week old male and female mice. Mice injected with DMEM were also used as controls. Mice were monitored daily for survival, weight loss, and disease symptoms to document the clinical course of viral infection. In the in vivo antiviral assay, 6-8 week old male and female mice were infected with 1000 PFU of SFTSV, HSV, or ZIKA. The AST-treated group was co-administered with 150 mg / kg / day of AST daily. The vehicle was used as a control, and the vehicle composition was 5% DMSO + 5% ethanol + 40% PEG400 + 50% saline. For tissue localization and pathological analysis, all mice were anesthetized, imaged, and tissues were collected at the indicated time points.

[0100] 2. Test results: We investigated whether AST could prevent and control SFTSV infection in mice. A preliminary study was conducted on A129 mice to determine whether AST pre-treatment and co-treatment showed similar disease progression as the control group. A129 mice aged 6 to 8 weeks were infected with SFTSV at an MOI of 5 via the intraperitoneal route and weight loss and mortality in these mice and mock-infected mice were monitored. By day 6, two of the five mice in the SFTSV-treated group had died, and all had died by day 7. Mice inoculated with the same dose of virus and co-treated with AST did not show typical clinical symptoms of the disease, and 60% of the group survived. Mice inoculated with the same dose of virus and pre-treated with AST did not show typical clinical symptoms of the disease, and 80% of the group survived. The results are as follows Figure 22 As shown, the survival rate of the blank control and astaxanthin-treated groups was 100%. We then performed immunohistochemistry and WB detection of NP protein in spleen tissue. The results showed that AST significantly inhibited the viral titer in mice. Figure 23 shown.

[0101] Observation of mice in all groups revealed that mice treated with SFTSV experienced significant weight loss, whereas those treated with AST maintained almost the same weight. However, HSV and ZIKA did not significantly alter mouse weight. Furthermore, pathological changes induced by HSV, SFTSV, and ZIKA infection in organs were assessed using H&E staining. In SFTSV-infected mice, the overall structure of spleen tissue was abnormal, with unclear demarcations between the white and red pulp, a dramatic decrease in red pulp, and unclear white pulp lymphoid nodule structure. Numerous lymphocytes were observed, with marked necrosis, prominent pyknosis, and fragmented nuclei, along with a small number of multinucleated giant cells. In mice treated with AST, the overall structure of spleen tissue was mildly abnormal, with clear demarcations between the red and white pulp, a well-defined splenic nodule structure, and no significant atrophy or reduction of splenic nodules. There was no significant connective tissue hyperplasia in the interstitial tissue, and a small number of multinucleated giant cell infiltration was observed. These results suggest that AST can reduce mortality and improve the outcome of viral infection in mice.

[0102] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, the present invention is intended to include such changes and modifications as fall within the scope of the claims and their equivalents.

Claims

1. The use of astaxanthin as the sole active ingredient in the preparation of an antiviral product, characterized in that: The virus is a coronavirus.

2. The use of astaxanthin as the sole active ingredient in the preparation of an antiviral product according to claim 1, characterized in that: The product is a drug.

3. The use of astaxanthin as the sole active ingredient in the preparation of an antiviral product according to claim 2, characterized in that: The astaxanthin is used for preparing medicine for preventing viral infection.

4. The use of astaxanthin as the sole active ingredient in the preparation of an antiviral product according to claim 2, characterized in that: The astaxanthin is used for preparing medicines for treating diseases caused by viral infections.

5. The use of astaxanthin as the sole active ingredient in the preparation of an antiviral product according to claim 2, characterized in that: The astaxanthin is mixed with a carrier or excipient to prepare a pharmaceutically acceptable dosage form.

6. The use of astaxanthin as the sole active ingredient in the preparation of an antiviral product according to claim 5, characterized in that: The medicine is an oral preparation or an injection preparation.

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

8. The use of astaxanthin as the sole active ingredient in the preparation of an antiviral product according to claim 6, characterized in that: The injection preparation is in the form of a solution or a suspension.

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 medicine is 1wt%-99.9wt%.

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