Application of astaxanthin in preparation of antiviral product

Abstract

The invention belongs to the technical field of antiviral drugs, and particularly relates to application of astaxanthin in preparation of antiviral products. The virus comprises a DNA virus, a positive-sense single-chain RNA virus and a single-strand negative-chain RNA virus. 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 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 far-reaching 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 fat and organic solvents, but insoluble in water. It exists naturally in some plants and animals as a red pigment, 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 remove ROS, thereby terminating chain oxidation reactions. The polymethylene olefin chain and terminal ring structure of astaxanthin increase the rigidity of the cell membrane and change the permeability of the cell membrane. Its polar end can pass through the cell membrane, thereby increasing the strength and mechanical stability of the cell membrane. Therefore, it can limit the entry of peroxide initiators into cells, protect important molecules in cells from oxidative damage, inhibit tumor occurrence, enhance the body's immunity, and also prevent eye diseases caused by diabetes.

[0004] There are currently no studies showing that astaxanthin can fight viral infections. Summary of the invention

[0005] Although some 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 is still a major medical need at this stage. The research results of the present invention show that the potential of astaxanthin as a new broad-spectrum antiviral small molecule compound has emerged. Therefore, the purpose of the present invention is to broaden the scope of efficacy of astaxanthin and provide its application in the preparation of antiviral products.

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

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

[0008] The present invention measures the transcription and expression levels of viruses in the human macrophage cell line THP-1, and the results show that astaxanthin can inhibit infection with 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 a 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 and a carrier or excipient are mixed 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 product. Currently, the astaxanthin health products on the market mainly include soft capsules, hard capsules and oral liquids. These products usually contain high-purity astaxanthin and are intended to provide consumers with antioxidant protection through oral administration. The health products provided by the present invention are intended to provide consumers with prevention of viral infections.

[0019] Beneficial effects of the present invention:

[0020] The present invention first discovered that astaxanthin can significantly 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, and further studies have shown that astaxanthin affects the mechanism of virus adsorption and internalization, and 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 the invasion of viruses 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 viral infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 middle:

[0022] A shows the results of RT-qPCR detection of extracellular and intracellular virus titers of 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.

[0023] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers of 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.

[0024] Figure 2 middle:

[0025] A shows the results of RT-qPCR detection of extracellular and intracellular virus titers of 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.

[0026] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers of 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.

[0027] Figure 3 middle:

[0028] 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.

[0029] 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.

[0030] Figure 4 middle:

[0031] A shows 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 treated with HcoV at an MOI of 1 for 24 hours.

[0032] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers of 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.

[0033] Figure 5 middle:

[0034] 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.

[0035] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers of 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.

[0036] Figure 6 middle:

[0037] A shows the results of RT-qPCR detection of extracellular and intracellular virus titers of 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.

[0038] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers of 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.

[0039] Figure 7 middle:

[0040] A shows the results of RT-qPCR detection of extracellular and intracellular virus titers of 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.

[0041] B shows the results of RT-qPCR detection of extracellular and intracellular virus titers of 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.

[0042] Figure 8 middle:

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

[0044] 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.

[0045] Fig. 9 middle:

[0046] 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.

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

[0048] Fig.10 middle:

[0049] 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 ZIKA at an MOI of 1 for 24 hours.

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

[0051] Fig.11 middle:

[0052] 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.

[0053] 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.

[0054] Fig.12 middle:

[0055] 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.

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

[0057] Fig.13 middle:

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

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

[0060] Fig.14 middle:

[0061] 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.

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

[0063] Fig.15 middle:

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

[0065] 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.

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

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

[0068] Fig.16 middle:

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

[0070] B shows the cellular Ca2+ of THP-1 cells after treatment with SFTSV and AST for 24 h. 2+ Flux.

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

[0072] Fig.17 middle:

[0073] A is the r value calculated by TMA-DPH probe, reflecting the effect of SFTSV on cell membrane fluidity at different times.

[0074] B shows the changes in membrane fluidity when cells were treated with SFTSV and 10 μM AST for 1 h.

[0075] Fig.18 middle:

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

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

[0078] Fig.19 middle:

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

[0080] B: Western blot detection of the expression level of clathrin.

[0081] Fig. 20 Western blot was used to detect the expression levels of all cell surface receptors in THP-1 cells treated with SFTSV and AST for 1 h.

[0082] Fig.21 middle:

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

[0084] B is a quantitative graph of actin expression level.

[0085] Fig. 22 Figure 2 is the Kaplan-Meier survival curve of A129 mice infected with SFTSV.

[0086] Fig.23 Western blot was used to detect the expression level of SFTSV NP protein in tissues. DETAILED DESCRIPTION

[0087] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but 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.

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

[0089] 1. Test materials:

[0090] SFTSV was a gift from Jiangsu Provincial Center for Disease Control and Prevention, see https: / / doi.org / 10.1080 / 15548627.2024.2356505.

[0091] Zika virus, ZIKA, was a gift from Jiangsu Provincial Center for Disease Control and Prevention.

[0092] Hand, foot and mouth virus, EV71, was a gift from Jiangsu Provincial Center for Disease Control and Prevention.

[0093] Herpes simplex virus, HSV, was gifted by Professor Zhong Bo from Wuhan University.

[0094] Human coronavirus, HcoV, was gifted by Professor Zhang Lei Ke of Wuhan Institute of Virology.

[0095] Influenza A virus, H1N1 was donated by Professor Zhang Lei Ke of Wuhan Institute of Virology.

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

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

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

[0099] Polyvinylidene fluoride membrane: Amersham, 10600023.

[0100] 2. Test methods:

[0101] 1. Cell culture:

[0102] 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% CO 2. Virus-infected cells and virus-infected and AST-cotreated cells were cultured in DMEM containing 2% fetal bovine serum and penicillin-streptomycin.

[0103] 2. Western Blotting:

[0104] Cells treated with AST and virus infection were collected and lysed with 1× SDS washing buffer supplemented with protease inhibitors. The extracted proteins were 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. The membranes were washed with TBST washing buffer containing 5% skim milk powder for 1 hour at room temperature and then incubated with appropriately diluted primary antibodies at 4°C overnight. The PVDF membranes were rinsed with TBST washing buffer three times for 5 minutes each and then incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour at a dilution ratio of 1:10000. The radioautographic signals were monitored on the ChemiDoc touch imaging system. β-actin was used as an internal control in the experiment.

[0105] 3. Confocal microscopy detection:

[0106] The treated cells were fixed with 4% methanol for 10 minutes at room temperature, and the fixed cell membranes were treated with 0.1% Triton X-100. 1 mL of permeabilization solution was added to each sample for 10 minutes. After permeabilization, 1 mL of PBS containing 2% BSA was used to block the non-specific binding of the antibody at room temperature for 30 minutes. The blocking solution was removed, 200 μL of the primary antibody diluted with PBS was added, and then the sample containing the primary antibody was placed in the chromatography cabinet for incubation at 4°C for 12-16 hours. After permeabilization, 1 mL of PBS containing 2% BSA was added to the sample and placed at room temperature for 30 minutes to block the non-specific binding of the antibody. The blocking solution was removed, 200 μL of the primary antibody diluted with PBS was added, and then the sample containing the primary antibody was incubated in the chromatography cabinet at 4°C for 12-16 hours. After the primary antibody incubation was completed, the primary antibody was removed and 1 mL of pre-cooled PBS was added to rinse the sample 3 times, 5 minutes each time. Dilute the fluorescent conjugated secondary antibody in PBS according to the dilution ratio, add 200 μL of secondary antibody to each sample, and incubate at room temperature in the dark for 30 minutes. Add 200 μL of nuclear stain DAPI and incubate at room temperature in the dark 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 1 mL of TRIzol to each cell, then blow the cells with a spray gun tip to complete the lysis, and transfer the cell lysate containing TRIzol to a 1.5 mL EP tube. Add 0.1 mL of chloroform substitute, cover the EP tube, and mix thoroughly on a vortex shaker at room temperature for 3 minutes. Centrifuge at 10,000g for 15 minutes at 4°C. After centrifugation, carefully remove the EP tube and leave the RNA mainly in the upper aqueous phase. Use a 200μL pipette tip to carefully transfer the upper aqueous phase into a new centrifuge tube, about 500μL of the upper aqueous phase, and do not transfer into the middle layer. After removing the upper aqueous phase, add 500μL of isopropanol, invert and mix, and leave at room temperature for 5 minutes. Then centrifuge the sample at 10,000g for 10 minutes and remove the supernatant. Add 1 mL of 75% ethanol made from DEPC water to each test tube, mix gently, cover the test tube, centrifuge at 7500g for 5 minutes, pour off the ethanol, dry the precipitate, and add 50 μL of DEPC water to dissolve the RNA. Genomic DNA removal and RNA reverse transcription were performed according to the HiScript III RT SuperMix for qPCR kit. A qPCR reaction system was established in a 96-well plate of an ABI fluorescent quantitative PCR instrument. The copy number of the target gene in the sample to be tested was determined by real-time detection of the intensity of the fluorescent signal that changes with amplification, and the Ct value was obtained.

[0109] 3. Test results:

[0110] 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. Figures 1 to 14 As shown in Figure 2, we found that AST could inhibit infection with 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 SFTSV infection led to a significant reduction in the accumulation of viral Np protein in THP-1 cells after the addition of AST, as shown in Figure 2. Fig.15 Overall, these results suggest that AST can be used as a broad-spectrum antiviral small molecule active substance.

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

[0112] 1. Test methods:

[0113] 1. Determination of plasma membrane potential:

[0114] 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, and then treated with AST and SFTSV for 30 minutes. After treatment, the waste liquid was discarded and the cells were rinsed three 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. Plasma membrane integrity detection:

[0116] LDH release from lactate dehydrogenase was used to assess plasma membrane integrity. Cells were seeded into six-well plates, adhered to the plate 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 4°C for 3 minutes at 250g, and 20 μL was aspirated into a transparent 96-well plate, and LDH detection solution was added. After the reaction, the absorbance value at 450nm was read on the SpectraMax i3x microplate reader; the total protein concentration of the cell precipitate was first detected with a BCA kit, and the average LDH value was calculated based on the total LDH value of each sample. The total protein concentration of the precipitate was first detected with a BCA kit, and the average LDH value was calculated based on the total LDH value of each sample, and the untreated group was used as t for normalization.

[0117] 3. Calcium ion Ca 2+ Flux determination:

[0118] Fluo-3 / AM calcium ion fluorescent probe is a membrane permeable probe. After entering the cytoplasm, it will be cleaved into Fluo-3 by intracellular esterases. Fluo-3 cannot pass through the cell membrane and therefore remains in the cell. The cells were evenly inoculated into a fluorescent 96-well plate, exposed to astaxanthin and SFTSV for 24 hours, and then incubated with 5μmol of Fluo-3 / AM probe at 37°C and weak light for 30 minutes; after loading the probe, the cells were rinsed 3 times with HBSS buffer, and finally 100μL of HBSS buffer was added to each well, and then incubated in a 37°C incubator for 30 minutes to ensure that the intracellular Fluo-3 / AM was completely converted to Fluo-3.

[0119] 4. Plasma membrane fluidity test:

[0120] Membrane fluidity is measured using the TMA-DPH probe. TMA-DPH is cylindrical and sensitive to reorientation caused by lipid interactions on the membrane. It is used for fluorescence polarization analysis in rotational motion studies. The fluorescence intensity of TMA-DPH itself is negative, and the fluorescence is enhanced 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 a 100 mm culture dish, adhered to the wall and treated with SFTSV and AST for 4 h; the cells were incubated with 5 μM TMA-DPH probe at 37 ° C in the dark for 20 min; the cells were collected, washed with HBSS buffer for 30 min, and then centrifuged for 3 min, keeping the cells moist as much as possible; before detection, the cells were transferred to a glass slide individually and detected by laser Raman spectroscopy with a polarizer. Before detection, the cells were transferred to a glass slide and detected by a laser Raman spectrometer with a polarizer, with an excitation wavelength of 325 nm and an emission wavelength of 430 nm. The fluorescence values ​​of the polarizer were calculated in the horizontal and vertical directions respectively.

[0122] 5. Cytoskeleton integrity detection:

[0123] The cell F-actin microfilaments were labeled with a phalloidin probe to observe the cytoskeleton and morphology. The cells were seeded on a confocal culture dish, adhered to the wall, treated with SFTSV and AST for 2 hours, rinsed three times with PBS, fixed with 4% paraformaldehyde for 15 minutes at room temperature, and rinsed three times with PBS for 5 minutes each time to ensure that there was no residual fixative; the cells were incubated with DAPI and phalloidin at room temperature for 15 minutes, and rinsed three times with PBS for 5 minutes each time; the confocal culture dish was placed under a laser confocal microscope for observation and photography. The confocal culture dish was placed under a laser confocal microscope for observation and photography.

[0124] 2. Test results:

[0125] 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 meshwork.

[0126] When cells were exposed to the virus, 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 Fig.16 As shown in A, this indicates that astaxanthin has a protective effect on membrane integrity.

[0127] Depolarization of the plasma membrane further leads to changes in membrane permeability and extracellular Ca 2+ Astaxanthin significantly inhibited the virus's damage to the integrity of the plasma membrane. The Fluo-4 / AM probe is a fluorescent indicator of the permeability of cells 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 Fig.16 As shown in B, this indicates that astaxanthin has a protective effect on membrane stability.

[0128] Plasma membrane potential is a key indicator of cell membrane integrity and stability. DiBAC4(3) is a potential-sensitive slow-response probe that can enter depolarized cells. Our assay results showed that when cells were exposed to the virus, the membrane potential changed compared to untreated cells with or without astaxanthin. Co-treatment with astaxanthin significantly alleviated the virus-induced membrane potential abnormalities, as shown in Figure 2. Fig.16 As shown in C.

[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 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. Fig.17 As shown in A, AST alone has the ability to reduce membrane fluidity, with r values ​​increasing from 0.25 ± 0.0082 to 0.34 ± 0.017 after AST exposure. Exposure to the virus increased membrane fluidity compared to untreated cells, with r values ​​decreasing from 0.25 ± 0.0082 to 0.11 ± 0.0047. Co-administration of AST restored r values ​​to near background levels, as shown in Fig.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 virus endocytosis process.

[0130] 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 Fig.18 Therefore, we harvested the protein 0.5 hours after adding the virus. The results of Western blotting showed that the expression of clathrin increased significantly after adding the virus, and the addition of astaxanthin significantly inhibited the increase of clathrin expression. The results are shown in Fig.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. Fig. 20 shown.

[0131] Microfilaments are widely present 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 is used to evaluate the effect of cell absorption of viruses on the skeleton. Fig.21As shown in A, in the control group and the group treated with AST alone, F-actin showed certain 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 co-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 Fig.21 As shown in B. The above results indicate that AST can reduce virus-induced F-actin assembly and phagocytosis.

[0132] 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.

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

[0134] 1. Test methods:

[0135] 1. Animal experiments:

[0136] 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, while mice injected with DMEM were used as controls. The survival rate, weight loss and disease symptoms of mice were monitored daily to record the clinical course of viral infection. In the in vivo antiviral test, 6-8 week old male and female mice were infected with 1000 PFU of SFTSV, HSV or ZIKA, and the AST treatment group was combined with 150 mg / kg / day of AST every day. 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 tissue collected at the designated time points.

[0137] 2. Test results:

[0138] We investigated whether AST could prevent and control mice from SFTSV infection. A preliminary study was conducted on A129 mice to determine whether AST pre-treatment and co-treatment would show similar disease progression as the control group. A129 mice aged 6 to 8 weeks were infected with SFTSV at an MOI = 5 by the intraperitoneal route and these mice and mock-infected mice were monitored for weight loss and mortality. By day 6, two of five mice in the SFTSV-treated group died, and all died by day 7. Mice inoculated with the same dose of virus and co-treated with AST did not show typical clinical signs of 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 signs of disease, and 80% of the group survived. The results are as follows Fig. 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 virus titer in mice. Fig.23 shown.

[0139] Observation of mice in all groups revealed that mice given SFTSV had a significant weight loss, while the weight of mice after AST remained almost unchanged, but HSV and ZIKA did not produce significant changes in the weight of mice. In addition, the pathological changes induced by HSV, SFTSV, and ZIKA infection in organs were evaluated by H&E staining. In mice infected with SFTSV, the overall structure of spleen tissue was abnormal, the boundary between white pulp and red pulp was unclear, the red pulp was sharply reduced, the structure of white pulp lymph nodules was unclear, more lymphocytes were visible, obvious necrosis, obvious condensation and fragmentation of cell nuclei, and a small number of multinucleated giant cells were visible. The overall structure of spleen tissue in mice treated with AST was slightly abnormal, the boundary between red pulp and white pulp was clear, the structure of spleen nodules was clear, no obvious atrophy and shrinkage of spleen nodules was observed, no obvious hyperplasia of connective tissue was observed in the interstitial tissue, and a small number of multinucleated giant cells were observed in the tissue. All these results indicate that AST can reduce the mortality rate and improve the outcome of viral infection in mice.

[0140] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Obviously, various changes and modifications may be made to the present invention by those skilled in the art without departing from the spirit and scope of the present invention. Thus, the present invention is intended to include such changes and modifications if they fall within the scope of the claims and their equivalents.

Claims

1. The use of astaxanthin in the preparation of antiviral products, characterized in that: The viruses include DNA viruses, positive-sense single-stranded RNA viruses and single-stranded negative-stranded 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-stranded RNA viruses include fever with thrombocytopenia syndrome virus and influenza A virus.

2. The use of astaxanthin in the preparation of antiviral products according to claim 1, characterized in that: The product described is a drug.

3. The use of astaxanthin in the preparation of antiviral products according to claim 2, characterized in that: The astaxanthin is used for preparing medicine for preventing viral infection.

4. The use of astaxanthin in the preparation of antiviral products according to claim 2, characterized in that: The astaxanthin is used for preparing medicines for treating diseases caused by viral infection.

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

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

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

8. The use of astaxanthin in the preparation of antiviral products 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 in the preparation of antiviral products according to claim 6, characterized in that: The content of astaxanthin in the medicine is 1wt%-99.9wt%.

10. The use of astaxanthin in the preparation of antiviral drugs according to claim 1, characterized in that: The product is a health product.

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