New application of hypericin in preparation of medicine for preventing or treating Zika virus infection

Abstract

The invention provides application of hypericin in preparation of a medicine for preventing or treating infection or diseases caused by Zika viruses, and further provides a medicine composition for preventing or treating infection or diseases caused by Zika viruses, and the medicine composition comprises effective dose of hypericin, at least one medicinal carrier and / or at least one medicinal auxiliary material. The hypericin is found to be capable of effectively inhibiting the Zika virus RNA replication effect and / or inhibiting the protein function (such as expression of envelope E protein) of the Zika virus for the first time, it is proved that the hypericin has the potential effect of preventing or treating infection or diseases caused by the Zika virus, and a new scheme is provided for preventing or treating the Zika virus. More potential use values of the hypericin can be explored, and a new direction is also provided for a scheme of anti-mosquito-borne virus drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a new medicinal use of a hypericin preparation for preventing or treating Zika virus infection. Background Art

[0002] Zika virus (ZIKV) was first discovered in rhesus monkeys in the Zika Forest of Uganda in 1947, hence its name. As of January 5, 2017, a total of 75 countries and regions around the world have reported outbreaks of Zika virus disease transmitted by mosquitoes, of which 58 countries and regions have reported for the first time since 2015.

[0003] Zika virus is a small enveloped positive-strand RNA virus, a mosquito-borne flavivirus, with an RNA genome encoding three structural genes (core C, membrane precursor prM and envelope E) and seven non-structural genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5), with the untranslated region (UTR) genes located at the 5' and 3' ends. The genomic RNA of the C protein contains a cell membrane-derived lipid bilayer shell with prM and envelope E proteins to form immature virus particles. The envelope E protein is the main protein involved in receptor binding and fusion, forming a head-to-tail dimer on the surface of the virus particle. The envelope E protein forms 90 dimers on the smooth surface of the mature virus. The non-structural proteins of Zika virus are involved in viral replication and assembly, mainly the cleavage of viral polyproteins, viral RNA replication and evasion of innate immune responses.

[0004] Zika virus is transmitted to humans through the bites of Aedes mosquitoes, causing Zika virus disease. Among people infected with Zika virus, 80% of them are asymptomatic, and only about 20% of the infected people have moderate self-limiting symptoms characterized by fever, rash, joint pain and conjunctivitis. However, studies have found that Zika virus infection is associated with microcephaly. In addition to microcephaly, Zika virus infection is also associated with other neurological diseases, including Guillain-Barré syndrome, encephalitis and myelitis. The most serious symptom at present is congenital Zika syndrome (CZS), which is characterized by microcephaly and other serious neurological complications caused by intrauterine infection.

[0005] In the past few decades, preventive vaccination as a means of preventing flavivirus infection has become an important research area of ​​concern. However, despite the huge prevalence and impact on the population, few flavivirus vaccines have been approved for humans so far. Because of the existence of cross-immunity and antibody-dependent enhancement effects, vaccine design needs to be protective against various related serotypes, making vaccine development difficult. Most of the mature vaccines that have been developed are inactivated vaccines or live attenuated vaccines with low efficiency. Due to the safety of children and people with immune deficiencies, the use of vaccines is limited. At present, symptomatic treatment is mainly adopted for diseases caused by Zika virus in clinical practice, and research on the discovery of antiviral drugs has been ongoing.

[0006] Hypericin (HYP) is a component first isolated from the plant Hypericum perforatum of the family Garcinia in 1957. It is the most biologically active substance naturally present in Hypericum perforatum and belongs to the dianthrone class of compounds with a chemical formula of C 30 H 16 O8, structural formula is shown below.

[0007]

[0008] Since the discovery of hypericin, people have conducted a lot of research on hypericin. Domestic research is mostly concentrated in the pharmacological clinical research stage, focusing on anti-tumor and antiviral effects. Foreign countries have used hypericin for clinical treatment of depression, AIDS, etc. At present, clinical trials have been conducted on cutaneous T-cell lymphoma plus mycosis fungoides (NCT05380635), HIV infection (NCT00000645, NCT00000792) and psoriasis, plaque psoriasis and psoriasis vulgaris (NCT05442190). So far, studies have reported that hypericin has a variety of pharmacological activities, such as anti-tumor, antibacterial and antidepressant activities, and can induce cell apoptosis. Hypericin has also been shown to have antiviral activity: anti-human immunodeficiency virus (HIV), anti-infective bronchitis virus (IBV), anti-hepatitis B virus (HBV), anti-herpes simplex virus type 1 (HSV-1), anti-new duck reovirus (NDRV) and other viruses. With the in-depth study of the activity of hypericin at home and abroad, hypericin has shown significant therapeutic potential in pharmacological effects and is a promising therapeutic drug. Many antiviral effects of hypericin have been reported, but its inhibitory effect on Zika virus is still unknown. Therefore, we used the widely reported Zika virus-infected BHK-21 cells as a model to test cell samples with different treatments to study whether hypericin has an inhibitory effect on Zika virus and reveal the antiviral activity of hypericin, which is conducive to exploring more potential use values ​​of hypericin and providing new directions for the scheme of anti-mosquito-borne virus drugs. Summary of the invention

[0009] Based on the problems existing in the above-mentioned prior art, after research, the present invention proposes the following technical solutions:

[0010] A first aspect of the present invention provides use of hypericin in the preparation of a medicament for preventing or treating an infection or disease caused by Zika virus.

[0011] In the present invention, the molecular formula of hypericin is C 30 H 16 O8, has the structural formula shown above.

[0012] Furthermore, the drug is used to inhibit the replication effect of Zika virus and / or inhibit the protein function of Zika virus.

[0013] Furthermore, the hypericin in the drug inhibits the expression of the envelope E protein of the Zika virus.

[0014] Furthermore, the medicine comprises an effective amount of hypericin.

[0015] Further, the drug is administered within 0-12 hours of infection or disease caused by Zika virus.

[0016] Furthermore, the drug is prepared from hypericin and at least one pharmaceutical carrier and / or at least one pharmaceutical excipient via a common pharmaceutical method.

[0017] Furthermore, the pharmaceutical excipients include at least one of a diluent, a binder, a disintegrant, a lubricant and a flavoring agent.

[0018] Furthermore, the drug is in the form of at least one of tablets, capsules, granules, injections and sprays.

[0019] Preferably, the administration method of the drug includes oral administration.

[0020] A second aspect of the present invention provides a pharmaceutical composition for preventing or treating infection or disease caused by Zika virus, comprising an effective amount of hypericin, at least one pharmaceutical carrier and / or at least one pharmaceutical excipient.

[0021] Furthermore, the pharmaceutical composition is administered at least 3 times per week.

[0022] In some embodiments, the effective concentration range of the cell experiment is 10 μmol / L-40 μmol / L, and the preferred concentration range is 10 μmol / L-20 μmol / L.

[0023] The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article. It should be noted that in the present invention, specific numerical values ​​can vary within ±10% as the numerical protection range of the present application.

[0024] In the description of the present invention, unless otherwise specified, "multiple / multiple" and other similar words mean two / kinds or more than two / kinds. In addition, "include", "including", "have", "contain" and any variations thereof used in the present invention are open terms, i.e., including but not limited to.

[0025] Unless otherwise indicated, all technical and scientific terms used in the present invention have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in the present invention are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of the present invention shall prevail.

[0026] The pharmaceutical composition of the present invention is used to prevent and / or treat infection or disease caused by Zika virus. In the present application, the terms "drug" and "pharmaceutical composition" can be used interchangeably, and "drug" or "pharmaceutical composition" refers to the preparation of a medium commonly used in the art for delivering biologically active compounds to mammals (such as humans). The purpose of the pharmaceutical composition is to promote the administration of an organism, facilitate the absorption of active ingredients and then exert biological activity. The term "pharmaceutical" used in the present invention refers to a substance (such as a pharmaceutical carrier or a pharmaceutical excipient) that does not affect the biological activity or properties of the compounds of the present invention, and is relatively non-toxic, that is, the substance can be applied to an individual without causing adverse biological reactions or interacting with any component contained in the composition in an adverse manner.

[0027] In the present invention, the pharmaceutical excipients include at least one of a diluent, a binder, a disintegrant, a lubricant and a flavoring agent.

[0028] In the present invention, at least one of the pharmaceutically acceptable carrier mixture.

[0029] In the present invention, the drug or drug composition can be made into various pharmaceutical preparations, including tablets (controlled release tablets, sustained release tablets), granules, capsules (hard capsules, soft capsules), pills, decoctions and other oral preparations.

[0030] In the present invention, the term "prevention" includes reducing the likelihood of a disease or condition occurring or worsening in a patient.

[0031] The term "treatment" and other similar synonyms in the present invention include the following meanings:

[0032] (i) preventing a disease or condition from occurring in a mammal, particularly where such mammal is susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition;

[0033] (ii) inhibiting a disease or condition, i.e. arresting its development;

[0034] (iii) alleviate a disease or condition, that is, cause the state of the disease or condition to regress;

[0035] (iv) alleviating the symptoms caused by the disease or condition.

[0036] In the present invention, the term "effective amount" or "pharmaceutically effective amount" refers to an amount that can produce a preventive or therapeutic effect on humans and / or other animals and can be accepted by humans and / or other animals. The result can be the reduction and / or alleviation of signs, symptoms or causes of disease, or any other desired changes in biological systems. For example, an "effective amount" for treatment is the amount of a composition comprising a compound disclosed herein that is required to provide a significant symptom alleviation effect clinically. Techniques such as dose escalation tests can be used to determine the effective amount suitable for any individual case.

[0037] In the present invention, the terms "administering", "administration" or "medication" are used interchangeably and refer to a method that can deliver a compound or composition to a desired site for biological action.

[0038] The drug or pharmaceutical composition of the present invention containing an effective amount of hypericin is used for mammals (including humans and other animals) that need to prevent or treat infections or diseases caused by Zika virus, wherein the dosage during administration is an effective dosage for prevention or treatment in medicine. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill range of skilled physicians.

[0039] The beneficial effects of the present invention include at least:

[0040] The present invention discovers for the first time that hypericin can effectively inhibit the RNA replication effect of Zika virus and / or inhibit the protein function of Zika virus (such as the expression of envelope E protein), proving that hypericin has the potential to prevent or treat infection or disease caused by Zika virus, providing a new solution for the prevention or treatment of Zika virus, which is conducive to exploring more potential use values ​​of hypericin, and also provides a new direction for the solution of anti-mosquito-borne virus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Showing the pathological features of C6 / 36 cells infected with Zika virus.

[0042] Figure 2 Shows the effect of different drug concentrations on cell survival rate, among which, Figure 2 Figure A is the hypericin treatment group. Figure 2 Figure B shows the curcumin-treated group.

[0043] Figure 3 Shown are the effects of hypericin on Zika virus RNA synthesis at different treatment times.

[0044] Figure 4 The inhibitory effect of hypericin on the release of Zika virus particles was observed at the cellular level using plaque assay.

[0045] Figure 5 Shown is the effect of adding drugs at different time points on Zika virus RNA synthesis.

[0046] Figure 6 Shows the effect of adding drugs at different time points on Zika virus RNA synthesis, among which, Figure 6 Figure A is the pretreatment group. Figure 6 Figure B shows the post-infection treatment group.

[0047] Figure 7 Hypericin has been shown to inhibit the expression of Zika virus envelope E protein, among which, Figure 7 Figure A in the figure is a western blot band of Zika virus envelope E protein. Figure 7 Figure B is a grayscale comparison of the expression of Zika virus envelope E protein.

[0048] Figure 8 Hypericin was shown to have an inhibitory effect on Zika virus at the cellular level using indirect immunofluorescence assay, where:

[0049] Group A: The hypericin-treated group had an inhibitory effect on the envelope E protein of Zika virus.

[0050] Panel B: Indirect immunofluorescence image of the Zika virus envelope E protein in the control group without drug treatment.

[0051] In order to further determine the inhibitory effect of hypericin on the envelope E protein of Zika virus, indirect immunofluorescence assay (IFA) was used to detect the inhibitory effect of hypericin on the envelope E protein of Zika virus. BHK-21 cells were infected with the virus and treated with 20 μmol / L hypericin. After 24 hours of culture, BHK-21 cells were collected and fixed, incubated with monoclonal antibodies to the envelope E protein of Zika virus and corresponding fluorescent secondary antibodies for the localization of the envelope E protein of Zika virus (green), and then stained with DAPI (blue). The results are shown in Figure 2. Figure 8 The green fluorescence intensity of the envelope E protein in BHK-21 cells treated with hypericin was weaker than that in the virus-treated group, indicating that the expression of the envelope E protein was inhibited.

[0052] Fig. 9 Shows the fluorescence images of Aedes albopictus fed with different concentrations of hypericin (HYP) in the midgut detected by inverted laser confocal microscopy.

[0053] Fig.10 Shown is the immunoblot detection of different concentrations of hypericin treatment in Aedes albopictus after oral infection with Zika virus. DETAILED DESCRIPTION

[0054] The present invention is further described below in conjunction with specific embodiments. It should be understood that the following contents are only used to explain the present invention and cannot be understood as limiting the present invention.

[0055] Without departing from the scope or spirit of the present invention, various modifications and variations may be made to the specific embodiments of the present invention description, which will be apparent to those skilled in the art. Other embodiments obtained from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only. The experimental methods in the following examples that do not specify specific conditions are generally tested under conventional conditions or under conditions recommended by the manufacturer.

[0056] Example 1

[0057] 1 Materials

[0058] 1.1 Compounds and reagents

[0059] Compounds and reagents Hypericin and curcumin were purchased from MedChemExpress, USA, fetal bovine serum (FBS) was purchased from Shanghai Jitai Yikesai Biological Co., Ltd., reverse transcription kit (GoScriptTM ReverseTranscription System, cat NO. A5001) was purchased from Promega, USA, fluorescent quantitative PCR kit (PowerGreen qPCR Mix ((Low ROX+, cat. NO. P2102a) was purchased from Guangzhou Dongsheng Biotechnology Co., Ltd., and primers were synthesized by Guangzhou Qingke Company.

[0060] 1.2 Cells and virus strains

[0061] Aedes albopictus cell line (C6 / 36 cells) and hamster kidney cells (BHK-21 cells) were cryopreserved in liquid nitrogen in our laboratory. Zika virus strain (GenBank: KU820899.2) was stored at -80°C in our laboratory.

[0062] 2 Methods

[0063] 2.1 Cell and virus culture

[0064] BHK-21 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). 5 U / L penicillin and streptomycin) were cultured in a 37°C, 5% CO2 incubator, and C6 / 36 cells were cultured in 1640 medium (containing 10 5 U / L penicillin and streptomycin) were cultured in an incubator at 28°C. Zika virus was enriched and amplified in C6 / 36 cells, and the virus titer was determined by plaque assay.

[0065] 2.2 MTT assay for cytotoxicity

[0066] BHK-21 cells in the logarithmic growth phase were inoculated into 96-well plates, and the cell density of each well of the cell suspension was adjusted to about 4000. 100 μl of cell suspension containing 10% DMEM FBS culture medium was added to each well, and the peripheral edge holes were filled with sterile PBS. After inoculation, the cells were placed in a 37°C, 5% CO2 incubator and cultured overnight until the cells adhered to the wall and grew to 80% to 90%, the culture medium was discarded, and PBS was washed. 100 μl of hypericin diluted to different concentrations (1.25, 2.5, 5, 10, 20, 40, 60, 80 μmol / L) was added to each well, and 4 replicates were set for each concentration, and a blank control group without cells and drugs and a negative control group without drugs were set. Place in 37 ℃, 5% CO2 incubator for 48 hours, add 90μl fresh serum-free DMEM medium to each well, add 10μl MTT solution, continue to culture for 4h, remove the culture medium in the well, add 110μl Formazan dissolving solution to each well, place on a shaker for 10min at low speed to fully dissolve the crystals. Measure the absorbance of each well at 490nm on an ELISA reader, take the average of 4 wells to calculate the cell survival rate (%), calculate the cell survival rate: the calculation formula is [A490 (drug treatment group) - A490 (blank control group)] ÷ [A490 (no drug treatment group) - A490 (blank control group)], the drug concentration and test method of the positive control drug curcumin cytotoxicity test are the same as hypericin.

[0067] 2.3 Effects of different drug concentrations on Zika virus replication

[0068] BHK-21 cells were plated in 12-well plates at 2 × 10 5 Cells were cultured overnight at 37°C and 5% CO2 incubator. When the cells grew to 80% density, the supernatant was discarded, and the cells were infected with a virus amount of MOI=5 for 1 hour. After the infection was completed, each well was washed twice with PBS, and then different concentrations (5, 10, 15, 20 μmol / L) of hypericin were added and a blank control group (BC) containing only cells and a positive control group (PC) containing cells and viruses were set at the same time. Three duplicate wells were set for each group, and each experiment was repeated three times. Cell supernatant, total cell RNA and total cell protein were collected 24 hours after the infection was completed, and the effect of hypericin on Zika virus RNA replication, expression of envelope E protein and virus particle formation was detected according to 2.5-2.7 method.

[0069] 2.4 Effects of adding drugs at different time points on viral replication

[0070] In order to explore the antiviral effect of hypericin, different drug treatment methods were used to study the antiviral effect of hypericin: (1) Pre-infection treatment, that is, BHK-21 cells were first treated with hypericin and then infected with the virus. BHK-21 cells in the logarithmic growth phase were inoculated into 12-well plates. After the cells grew to 80%-90% of the cells, the original culture medium was discarded and rinsed with PBS. 1 ml of 2% FBSDMEM culture medium containing double antibodies and different hypericin drug concentrations (10, 20 μmol / L) was replaced in each well, and a positive control group with virus and no drug and a negative control group without virus and drug were set up. Three replicate wells were set for each treatment group. After incubation in a 37°C, 5% CO2 incubator for 2 hours, BHK-21 cells were infected with Zika virus at an MOI of 5, incubated in a 37°C, 5% CO2 incubator for 1 hour, rinsed twice with PBS, 1 ml of fresh 2% FBSDMEM culture medium containing double antibodies was added to each well, and cultured in a 37°C, 5% CO2 incubator for 24 hours, and samples were collected. (2) Post-infection treatment, i.e., BHK-21 cells were treated with drugs after being infected with the virus. After BHK-21 cells were inoculated into 12-well plates and grown to 80%-90%, the BHK-21 cells were infected with Zika virus at an MOI of 5, and incubated in a 37°C, 5% CO2 incubator for 1 hour. Hypericin at a concentration of 10 μmol / L was added at 0, 2, 4, 8, 12, 24, and 36 hours, respectively. A positive control group with virus and no drug and a negative control group without virus and drug were set up. Three replicate wells were set up for each treatment group. After the treatment, the cells were placed in a 37°C, 5% CO2 incubator for 48 hours, and the samples were collected.

[0071] 2.5 Real-time fluorescence quantitative PCR detection of drug effects on ZIKV RNA replication

[0072] Cell RNA was extracted using the Trizol method, and qRT-PCR detection was performed after RNA reverse transcription. The qRT-PCR detection reaction system (20ml): 2×Power Green Qpcr Mix 10ml, 0.4ml of upstream and downstream primers (10μM), cDNA 2ml, ddH2O 7.2ml. Reaction conditions: 94℃ pre-denaturation for 3min; 94℃, 15s; 55℃, 15s; 72℃, 20s; 40 cycles in total. The amplified target gene is the 3'-UTR region of the ZIKV virus. Upstream primer: 5'-CTATAGTCAGGCCGAGAACGC', lower primer: 5'-CCCAGATTAAAGGGTGGGGA-3'. According to the amplification results, a dose-response curve was compiled to calculate the half effective inhibitory concentration (50% effective concentration, EC 50 )

[0073] 2.6 Western blot to detect the inhibitory effect of drugs on the expression of Zika virus envelope E protein

[0074] After extracting total cell protein, 10% SDS-PAGE electrophoresis gel was used to separate the target protein and internal reference protein, and the separated gel was transferred to a PVDF membrane, blocked with 5% BSA at 37°C for 2h, and primary antibodies of Zika virus envelope E protein antibody (1:5000) and GAPDH antibody (1:1000) were added respectively, incubated overnight at 4°C shaker, and washed three times with PBST; horseradish peroxidase-labeled secondary antibody (1:5000) was added and incubated at 37°C for 2h, washed three times with PBST, and developed with ECL luminescent solution. Grayscale values ​​were analyzed using Image-J software to calculate the relative expression of envelope E protein.

[0075] 2.7 Plaque assay to determine virus titer in cell supernatant

[0076] BHK-21 cells were seeded in 12-well plates at 2 × 10 5 cells, cultured overnight at 37°C, 5% CO2. When the cells were fully grown, the frozen drug-treated cell supernatant was taken out, and after gradient dilution, 400 ml of the diluted supernatant was added to each well, and a negative control group with only 2% DMEM culture medium was set up, and 2 replicate wells were set up for each concentration. The cells were shaken on a shaker for 15 min, cultured for 2 h at 37°C, 5% CO2, and the supernatant was discarded. The cells were washed twice with PBS, 1 ml of 1.5% methylcellulose was added to each well, and cultured at 37°C, 5% CO2 until the sixth day. 1 ml of 4% paraformaldehyde was added to each well and fixed at room temperature for 1 h, and the methylcellulose and paraformaldehyde were discarded. After washing, 200 ml of 0.1% crystal violet stain was added to each well, incubated at room temperature for 15 min, washed with clean water, and the plaques were counted and photographed.

[0077] 2.8 Verification of the expression of Zika virus envelope E protein by indirect immunofluorescence assay

[0078] BHK-21 cells were plated in a 12-well plate with a cell slide, with 2×10 5Cells were cultured overnight at 37°C and 5% CO2 incubator. When the cells grew to 80% density, the supernatant was discarded and the cells were infected with a virus at MOI = 5 for 1 hour. After infection, each well was washed twice with PBS, and then hypericin at a concentration of 20μmol / L was added. After 24 hours of culture, the culture medium was discarded, 1ml of PBS was added to each well, and the cells were rinsed at a low speed on a shaker for 5 minutes each time, and repeated 3 times. 1ml of 4% paraformaldehyde was added to each well, and the 12-well plate was placed at room temperature for 30 minutes. After fixation, 1ml of PBS was added to each well, and the cells were rinsed at a low speed on a shaker for 5 minutes each time, and repeated 3 times. Then 300ml of 0.1% Triton tissue permeabilization solution was added to each well, and the cells were left standing at room temperature for 20 minutes, and the solution was discarded. The PBS was washed three times, each time for 5 minutes. Add 1 ml of 10% BSA to each well, block at room temperature for 2 hours, add ZIKV protein E antibody (1:5000) and incubate overnight at 4℃ shaker, wash with PBS; add green fluorescent secondary antibody (1:1000) and incubate at 37℃ for 1 hour, wash with PBS. Take out the slide, wash once with pure water, put it on filter paper to dry in the dark, and seal it on a slide with a sealing agent containing DAPI. Observe and take pictures with an upright laser confocal microscope.

[0079] 2.9 Aedes albopictus infected with Zika virus

[0080] Aedes albopictus were raised at a constant 28°C and 70% relative humidity with a 12-hour light-dark cycle. After hatching, the larvae were transferred to a plastic container filled with clean water and fed with turtle food as food every day. Subsequently, the pupae were collected and transferred to a special insect culture container. Adult mosquitoes were fed with cotton balls soaked in 10% sucrose solution. Mosquito infection with Zika virus: After 12 hours of sugar deprivation from newly hatched 3-5-day-old Aedes albopictus female mosquitoes, the experimental group of mosquitoes were fed with defibrinated sheep blood mixed with Zika virus supernatant (ratio of 2:1). The control group of mosquitoes were fed with defibrinated sheep blood mixed with cell culture supernatant. After the blood meal, each mosquito was examined by stereomicroscope, and the blood-satiated mosquitoes were detected. These mosquitoes were maintained in the above culture conditions and the mosquito culture was continued.

[0081] 2.10 Data Processing

[0082] The experimental data were analyzed and processed using SPSS21.0 software, and the pictures were drawn using GraphPad Prism.

[0083] 3 Results

[0084] Pathological effects of 3.1C6 / 36 cells infected with Zika virus

[0085] After C6 / 36 cells were infected with Zika virus, they were placed in a 37°C incubator and the growth and pathological changes of the cells were observed under a low power microscope every day. The uninfected normal cells (control group) were spherical in shape. There was no obvious change in cell morphology 1-5 days after infection with the virus. Figure 1 shown.

[0086] 3.2 Cytotoxicity of hypericin

[0087] BHK-21 cells in logarithmic growth phase were inoculated into 96-well plates, and the cell density per well of the cell suspension was adjusted to about 4000. 100 μl of cell suspension containing 10% DMEM FBS culture medium was added to each well, and the peripheral edge wells were filled with sterile PBS. After inoculation, the cells were placed in a 37°C, 5% CO2 incubator and cultured overnight until the cells adhered to the wall and grew to 80%-90%, the culture medium was discarded, and PBS was washed. 100 μl of hypericin diluted to different concentrations (1.25, 2.5, 5, 10, 20, 40, 60, 80 μmol / L) was added to each well, and 4 replicates were set for each concentration, and a blank control group without cells and drugs and a negative control group without drugs were set. Place in a 37°C, 5% CO2 incubator for 48 hours, add 90 μl of fresh serum-free DMEM medium to each well, then add 10 μl of MTT solution, continue to culture for 4 hours, remove the culture medium in the well, add 110 μl of Formazan solution to each well, place on a shaker and shake at low speed for 10 minutes to fully dissolve the crystals. Measure the absorbance of each well at 490 nm on an ELISA reader, take the average of the 4 wells to calculate the cell survival rate (%), calculate the cell survival rate: the calculation formula is [A490 (drug treatment group) - A490 (blank control group)] ÷ [A490 (no drug treatment group) - A490 (blank control group)], the drug concentration and test method of the positive control drug curcumin cytotoxicity test are the same as hypericin, such as Figure 2 shown.

[0088] 3.3 Hypericin inhibits Zika virus (ZIKV) RNA replication

[0089] (1) BHK-21 cells were plated in 12-well plates at 2×10 cells per well. 5Cells were cultured overnight at 37°C and 5% CO2 incubator. When the cells grew to 80% density, the supernatant was discarded and the cells were infected with a virus at an MOI of 5 for 1 hour. After the infection was completed, each well was washed twice with PBS, and then hypericin at a concentration of 10 μmol / L was added. Total cell RNA was collected 12, 24, and 48 hours after the infection was completed to detect the effect of hypericin at different times on Zika virus RNA expression. The results showed that each drug treatment group with hypericin acting for 12 hours, 24 hours, and 48 hours could inhibit Zika virus RNA replication (P < 0.05). Figure 3 shown.

[0090] (2) Plaque assay to determine virus titer in cell supernatant

[0091] BHK-21 cells were seeded in 12-well plates at 2 × 10 5 cells, cultured overnight at 37°C, 5% CO2, and when the cells were full, the frozen drug-treated cell supernatant was taken out, and after gradient dilution, 400 μl of the diluted supernatant was added to each well, and a negative control group with only 2% DMEM culture medium was set up, and two replicate wells were set up for each concentration, shaken on a shaker for 15 minutes, cultured for 2 hours at 37°C, 5% CO2, and the supernatant was discarded. PBS was washed twice, 1 ml of 1.5% methylcellulose was added to each well, and cultured at 37°C, 5% CO2 until the sixth day. 1 ml of 4% paraformaldehyde was added to each well and fixed at room temperature for 1 hour, and the methylcellulose and paraformaldehyde were discarded. After washing, 200 μl of 0.1% crystal violet staining was added to each well, incubated at room temperature for 15 minutes, washed with water, and the plaques were counted and photographed. The effect of hypericin on the synthesis of complete virions of Zika virus was detected by the plaque method. Three repeated independent experiments showed that hypericin had an inhibitory effect on the synthesis of complete virions of Zika virus, 10 -1 When diluted, no plaques appeared in the hypericin drug treatment groups at 5, 10, 15, and 20 μmol / L concentrations, and the virus titer of the virus control group was 1.825×10 4 PFU / ml, such as Figure 4 shown.

[0092] 3.4 Effects of different hypericin concentrations on Zika virus RNA

[0093] After infection with Zika virus, the virus-infected cells were treated with different concentrations of hypericin or curcumin. After 24 hours of culture, the cells were collected to detect the inhibitory effect of hypericin or curcumin on Zika virus RNA. The results showed that hypericin inhibited the number of Zika virus RNA copies in a dose-dependent manner. Compared with the virus control group, the copy number of the hypericin treatment groups at concentrations of 5, 10, 15, and 20 μmol / L decreased by (58.94±14.71)%, (68.31±13.00)%, (79.64±7.08)%, and (83.95±6.01)%, respectively. The inhibitory effect was statistically significant at a concentration of 5 μmol / L. Compared with the virus group, the copy number of Zika virus RNA in the curcumin treatment group was reduced by (60.76±24.78)%, (73.81±13.60)%, (74.33±29.50)%, and (87.21±5.50)%, respectively. The inhibitory effect was statistically significant at a concentration of 5 μmol / L (P<0.01). 50 The inhibitory effect of hypericin on Zika virus RNA was 3.544 μmol / L. Real-time fluorescent quantitative PCR (qPCR) was used to detect the inhibitory effect of hypericin on Zika virus RNA copy number. The results showed that the inhibitory effect of hypericin on Zika virus RNA copy number was dose-dependent, and the differences between each drug treatment group and the virus group were statistically significant (P < 0.05). Figure 5 shown.

[0094] 3.5 Effects of adding drugs at different time points on Zika virus RNA

[0095] To study when hypericin is best added, two different ways of administration were used to study its effect on Zika virus RNA in BHK-21 cells. (1) Add the drug before infection. When BHK-21 cells grow to 80%-90%, incubate the cells with different doses of hypericin (10μmol / L, 20μmol / L) for 2 hours, then infect the cells with Zika virus for 1 hour and culture for 24 hours. (2) BHK-21 cells were plated in 12-well plates and when the cells grew to a density of 80%-90%, the cells were infected with a virus at an MOI of 5 for 1 hour. After infection, each well was washed twice with PBS. Then, 10 μmol / L hypericin was added at 0, 2, 4, 8, 12, 24, and 36 hours after infection. Cell samples were collected 48 hours after infection to detect the effect of hypericin on Zika virus RNA expression at different action times. The results showed that the addition of 10 μmol / L and 20 μmol / L hypericin before infection both had an inhibitory effect on Zika virus RNA synthesis. The 10 μmol / L and 20 μmol / L hypericin treatment groups were statistically significant compared with the virus group. Figure 6 As shown in Figure A. Adding 10μmol / L hypericin at different time points after infection had an effect on RNA synthesis. The inhibitory effect of adding hypericin within 0-12 hours was the best. The effect was weakened when adding hypericin 24 hours after infection. When adding hypericin 36 hours after infection, the synthesis of Zika virus RNA was no longer statistically different from that of the virus group. Figure 6 As shown in Figure B.

[0096] 3.6 Study on the mechanism of hypericin inhibiting Zika virus replication

[0097] (1) Western blot to detect the inhibitory effect of drugs on the expression of Zika virus envelope E protein

[0098] In order to explore the effect of different concentrations of hypericin on the envelope E protein of Zika virus, when BHK-21 cells grew to 80%-90%, ZIKV infected the cells with MOI=5 for 1 hour, treated with different doses of hypericin (5, 10, 15, 20 μmol / L), cultured for 24 hours, and collected the total intracellular protein. After extracting the total cell protein, 10% SDS-PAGE electrophoresis gel was used to separate the target protein and the internal reference protein, and the separated gel was transferred to a PVDF membrane, blocked with 5% BSA at 37°C for 2h, and Zika virus envelope E protein antibody (1:5000) and GAPDH antibody (1:1000) were added as primary antibodies, incubated overnight at 4°C on a shaker, and washed three times with PBST; horseradish peroxidase-labeled secondary antibody (1:5000) was added and incubated at 37°C for 2h, the membrane was washed three times with PBST, and developed with ECL luminescent solution. Image-J software was used to analyze the gray value and calculate the relative expression of envelope E protein.

[0099] The total protein of BHK-21 cells after Zika virus infection and drug treatment was extracted for Western blot detection. The results showed that hypericin could inhibit the expression of Zika virus envelope E protein. The expression of envelope E protein decreased with the increase of drug concentration. There were statistical differences between each concentration group and the virus group (P < 0.05). Figure 7 shown.

[0100] (2) Indirect immunofluorescence method to verify the expression of Zika virus envelope protein E

[0101] In order to further determine the inhibitory effect of hypericin on the envelope E protein of Zika virus, indirect immunofluorescence (IFA) was used to detect the inhibitory effect of hypericin on the envelope E protein of Zika virus.

[0102] BHK-21 cells were plated in a 12-well plate with a cell slide, with 2×10 5Cells were cultured overnight at 37°C and 5% CO2 incubator. When the cells grew to 80% density, the supernatant was discarded and the cells were infected with a virus at MOI = 5 for 1 hour. After infection, each well was washed twice with PBS, and then hypericin at a concentration of 20μmol / L was added. After 24 hours of culture, the culture medium was discarded, 1ml of PBS was added to each well, and the cells were rinsed at a low speed on a shaker for 5 minutes each time, and repeated 3 times. 1ml of 4% paraformaldehyde was added to each well, and the 12-well plate was placed at room temperature for 30 minutes. After fixation, 1ml of PBS was added to each well, and the cells were rinsed at a low speed on a shaker for 5 minutes each time, and repeated 3 times. Then 300μl of Triton (0.1% TritonX-100) was added to each well, and the cells were left at room temperature for 20 minutes, and the liquid was discarded. The PBS was washed three times, each time for 5 minutes. Add 1 ml of 10% BSA to each well, block at room temperature for 2 hours, add ZIKV protein E antibody (1:5000) and incubate overnight at 4℃ shaker, wash with PBS; add green fluorescent secondary antibody (1:1000) and incubate at 37℃ for 1 hour, wash with PBS. Take out the slide, wash it with pure water once, put it on filter paper to dry in the dark, and seal it on a slide with a sealing agent containing DAPI. Observe and take pictures with an upright laser confocal microscope.

[0103] It was found that after infection with the virus, the cells were treated with 20 μmol / L hypericin and tested after 24 hours of culture. After fixation, the BHK-21 cells were incubated with the monoclonal antibody of the Zika virus envelope E protein and the corresponding fluorescent secondary antibody. The localization of the Zika virus envelope E protein was detected with the fluorescent secondary antibody (green), and the cell nucleus was stained with DAPI (blue). From the results of IFA, it can be clearly observed that the green fluorescence intensity of the envelope E protein after hypericin treatment is weaker than that of the virus treatment group, such as Figure 8 shown.

[0104] 4. Observation of the inhibitory effect of hypericin on Zika virus replication using the Aedes albopictus model organism

[0105] Aedes albopictus is the main vector of Zika virus. The effect of hypericin on inhibiting the replication of Zika virus was observed using Aedes albopictus as a model organism. Hypericin was diluted with 10% glucose to prepare drug solutions with a Hypericin concentration of 10μmol / L and 40μmol / L, respectively. In order to promote Aedes albopictus to ingest the drug, a small amount of honey was added to the 10% glucose solution. The blood-saturated mosquitoes that ingested the Zika virus were transferred to a cylindrical container with a nylon mesh. After 12 hours of virus infection, the prepared drug was provided to the mosquitoes. 24 hours after ingesting hypericin, the mosquito midgut was dissected, and the ingestion of hypericin by Aedes albopictus was observed using an inverted laser confocal microscope. The results showed that the intensity of the fluorescence parameter was higher than the fluorescence signal of 40μmol / L hypericin (such as Fig. 9 The same parameters were used in the control group, and no fluorescence signal was detected (as shown in Figure C). Fig. 9Different concentrations of hypericin were provided to Aedes albopictus for feeding, and the results showed that the fluorescence signal of the control group was undetectable (as shown in Figure A and Figure B). Fig. 9 D in Figure ), the same parameters were used to detect 10μmol / L HYP and 40μmol / L HYP, and the results showed that the strong fluorescence signal increased in a gradient manner (as shown in Figure D in Figure ). Fig. 9 The results of Western blot experiments showed that hypericin inhibited the expression of Zika virus envelope E protein in Aedes albopictus, and there was a dose-response relationship (as shown in Figures E and F in Figure 1). Fig.10 shown).

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. Use of hypericin in the preparation of a medicament for preventing or treating an infection or disease caused by Zika virus.

2. The use according to claim 1, characterized in that The drug is used to inhibit the replication effect of Zika virus and / or inhibit the protein function of Zika virus.

3. The use according to claim 2, characterized in that: The hypericin in the drug inhibits the expression of the envelope E protein of the Zika virus.

4. The use according to any one of claims 1 to 3, characterized in that The medicament comprises an effective amount of hypericin.

5. The use according to claim 4, characterized in that Administer within 0-12 hours of Zika virus infection or disease.

6. The use according to claim 4, characterized in that The medicine is prepared from hypericin and at least one pharmaceutical carrier and / or at least one pharmaceutical excipient through a common pharmaceutical method.

7. The use according to claim 4, characterized in that The pharmaceutical excipients include at least one of a diluent, a binder, a disintegrant, a lubricant and a flavoring agent; and / or the dosage form of the drug includes at least one of a tablet, a capsule, a granule, an injection and a spray.

8. The use according to claim 4, characterized in that The administration method of the drug includes oral administration or injection administration.

9. A pharmaceutical composition for preventing or treating infection or disease caused by Zika virus, characterized in that: The invention comprises an effective amount of hypericin, at least one pharmaceutical carrier and / or at least one pharmaceutical excipient.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition is administered at least 3 times per week.

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