Application of Alloimperatorin in preparation of antiviral drugs

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of Alloimperatorin in preparation of an antiviral drug. It is found and verified for the first time that the Alloimperatorin can effectively inhibit replication of various viruses including bovine parainfluenza virus III, sendai virus, vesicular stomatitis virus, bovine ephemeral fever virus and herpes simplex virus I, virus release is blocked, toxicity to cells is relatively small, it is indicated that the Alloimperatorin has the prospect of being developed into broad-spectrum antiviral drugs, and the Alloimperatorin has good application prospects. A new medicinal application is developed for the Alloimperatorin, an experimental foundation is also laid for developing efficient and specific antiviral drugs, and the Alloimperatorin has good application value.

Description

Technical Field

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

[0002] At present, most of the research and development of antiviral drugs are virus enzyme inhibitor drugs. Such drugs have the advantages of clear targets, high specificity, and strong efficacy. However, they have a narrow antiviral spectrum and are prone to drug resistance. Therefore, screening candidate target molecules involved in virus replication and screening antiviral drugs with new antiviral mechanisms and broad-spectrum antiviral activities are urgent clinical needs.

[0003] Alloimperatorin, also known as Prangenidin, has a Chinese name of alloimperatorin, its CAS number is 642-05-7, and its molecular formula is C 16 H 14 O4. Alloimperatorin is a coumarin compound extracted from Angelica dahurica, and has antitumor activity. In HL-60 acute myeloid leukemia cancer cells, it can play an in vitro antitumor role by inducing apoptosis, disrupting the cell cycle, and inhibiting cell migration. However, there is no reported application of Alloimperatorin in the treatment of various diseases or conditions caused by viruses. Summary of the Invention

[0004] Aiming at the deficiencies of the existing technology, the present invention provides the application of Alloimperatorin in the preparation of antiviral drugs. The present invention discovers and confirms for the first time through research that Alloimperatorin has the effects against bovine parainfluenza virus type III, Sendai virus, vesicular stomatitis virus, bovine epizootic fever virus, and herpes simplex virus type I. Therefore, it has the prospect of being developed into an antiviral drug. Based on the above research results, the present invention is completed.

[0005] To achieve the above object, the present invention provides the following technical solutions: In the first aspect of the present invention, there is provided the application of Alloimperatorin in the preparation of antiviral products; wherein, the virus includes at least one of bovine parainfluenza virus type III, Sendai virus, vesicular stomatitis virus, bovine epizootic fever virus, and herpes simplex virus type I.

[0006] In the second aspect of the present invention, there is provided the application of Alloimperatorin in the preparation of products for inactivating viruses.

[0007] In the third aspect of the present invention, there is provided the use of Alloimperatorin in the preparation of a product for inhibiting virus replication.

[0008] In the fourth aspect of the present invention, there is provided a pharmaceutical composition, which is composed of Alloimperatorin and at least one other pharmaceutically active ingredient and / or at least one other non-pharmaceutically active ingredient.

[0009] The beneficial effects of the above one or more technical solutions are as follows: (1) The present invention discovers for the first time that Alloimperatorin has the effect of inhibiting in vitro virus infection, providing a new idea for the preparation of antiviral drugs.

[0010] (2) By comprehensively using techniques such as cell biology and virology, the present invention discovers and verifies for the first time that Alloimperatorin can effectively inhibit the replication of a variety of viruses including bovine parainfluenza virus type Ⅲ, Sendai virus, vesicular stomatitis virus, bovine ephemeral fever virus, and herpes simplex virus type I, block virus release, and has relatively low toxicity to cells, indicating its prospect of being developed into a broad-spectrum antiviral drug, opening up a new drug use for Alloimperatorin, and also laying an experimental foundation for the development of highly efficient and specific antiviral drugs, with good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0012] Figure 1 CC50 of Alloimperatorin in HEK293 cells, BHK-21 cells, and MDBK cells in the embodiments of the present invention; Figure 2 IC of Alloimperatorin against BPIV3, SeV, VSV, HSV-1, and BEFV viruses in the embodiments of the present invention 50 value; Figure 3 Effect of Alloimperatorin on inhibiting the replication of BPIV3, SeV, VSV, HSV-1, and BEFV in the embodiments of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0013] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0014] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0015] The present invention will be further described in conjunction with specific examples below. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by reagent companies; for the reagents, consumables, etc. used in the following examples, unless otherwise specified, the actual, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field and can be obtained from commercial channels.

[0016] As mentioned above, the application of Alloimperatorin in antiviral aspects has not been found yet.

[0017] In view of this, in a typical embodiment of the present invention, there is provided the application of Alloimperatorin in the preparation of antiviral products; wherein, the virus includes at least one of bovine parainfluenza virus type III, Sendai virus, vesicular stomatitis virus, bovine epizootic fever virus, and herpes simplex virus type I.

[0018] The full name of the Alloimperatorin is 9-hydroxy-4-(3-methyl-2-buten-1-yl)-7H-furo[3,2-g][1]benzopyran-7-one, its CAS number is 642-05-7, and its structural formula is: 。

[0019] It should be noted that this application is disclosed for the first time and is different from its known drug uses.

[0020] The application of the Alloimperatorin as an active ingredient or one of the active ingredients in the preparation of antiviral products; preferably, the concentration of the Alloimperatorin is 50 μg / mL.

[0021] The antiviral activity at least shows the ability to directly inactivate the above viruses and / or inhibit the replication of the above viruses.

[0022] The product can be a drug or a test reagent, and the test reagent is used for basic research and can thus be used to construct relevant cell or animal models.

[0023] Therefore, in another specific embodiment of the present invention, provided is: (a)The application of Alloimperatorin in the preparation of products for inactivating viruses; (b)The application of Alloimperatorin in the preparation of products for inhibiting virus replication.

[0024] Wherein, the virus includes at least one of bovine parainfluenza virus type III, Sendai virus, vesicular stomatitis virus, bovine epizootic fever virus, and herpes simplex virus type I.

[0025] According to the present invention, not only the application of Alloimperatorin in the preparation of antiviral drugs is disclosed, but also it is disclosed that when Alloimperatorin is administered in combination with at least one other pharmaceutically active ingredient, this effect can be enhanced. As an alternative or supplement to other pharmaceutically active ingredients, Alloimperatorin can also be used in combination with other non-pharmaceutically active ingredients.

[0026] In another specific embodiment of the present invention, a pharmaceutical composition is provided, and the composition is composed of Alloimperatorin and at least one other pharmaceutically active ingredient and / or at least one other non-pharmaceutically active ingredient.

[0027] The other pharmaceutically active ingredients include substances that can inhibit and / or kill the above-mentioned viruses.

[0028] The non-pharmaceutically active ingredients include pharmaceutically acceptable carriers, excipients, and / or diluents.

[0029] In another specific embodiment of the present invention, the non-pharmaceutically active ingredients include: Pharmaceutically compatible inorganic or organic acids or bases, polymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants, or lipids; Pharmacologically harmless salts, flavoring agents, vitamins, antioxidants, as well as stabilizers and / or preservatives.

[0030] The dosage forms of the pharmaceutical composition include: liquid dosage forms, solid dosage forms, topical preparations, and sprays.

[0031] In another specific embodiment of the present invention, the following dosage forms are included: true solution types, colloid types, microparticle dosage forms, emulsion dosage forms, suspension dosage forms, tablets, capsules, dripping pills, aerosols, pills, powders, solution agents, mixed suspension dosage forms, emulsions, granules, suppositories, lyophilized powder injections, inclusion compounds, landfill agents, patches, liniments.

[0032] Examples 1. Materials Human embryonic kidney cells (HEK293), hamster kidney fibroblasts (BHK-21), bovine kidney fibroblasts (MDBK); Bovine parainfluenza virus type 3 (BPIV3), Bovine Ephemeral Fever Virus (BEFV), Sendai virus (SeV), Vesicular Stomatitis Virus (VSV), Herpes simplex virus type 1 (HSV-1); The above cells and viruses are all stored in the Ruminant Disease Research Center of Shandong Normal University. Alloimperatorin was purchased from TargetMOI, DMSO was purchased from Sigma, and cell culture reagents such as DMEM medium, PBS, and fetal bovine serum were purchased from Vivacell Company.

[0033] 2. Methods and Results 2.1 Cytotoxicity Detection of Alloimperatorin The CCK-8 kit was used to detect the cytotoxic effects of Alloimperatorin on HEK293 cells, BHK-21 cells, and MDBK cells. In a 10 cm dish, the above cells that had grown into a monolayer were digested with trypsin to disperse the cells, and the cell count was taken. 100 μL of cell suspension (5×10 5 cells / well) was inoculated into a 96-well culture plate. The culture plate was placed in a 5% CO2 cell culture incubator at 37 °C for 12 - 24 h. After observing that the cells adhered well, the medium in each well was aspirated. In the experimental group, DMEM medium containing different concentrations of Alloimperatorin (0, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100 μg / mL) was added, and in the blank group, normal medium was added. Then, the 96-well plate was incubated in a 5% CO2 incubator at 37 °C for 48 h. 10 μL of CCK-8 solution was directly added to each well and incubated in a 5% CO2 incubator at 37 °C for 1 h. The 96-well plate was taken out, and the OD values of each well at a wavelength of 450 nm were measured with an enzyme-linked immunosorbent assay (ELISA) reader. The data were analyzed and processed, and the cell survival rate was calculated: Cell survival rate % = (OD of drug-treated cells / OD of control cells) × 100%. The drug concentration (CC 50 ) when T / C = 50% was calculated and a proliferation curve was plotted.

[0034] The results showed that the CC of Alloimperatorin in HEK293 cells, BHK-21 cells, and MDBK cells 50were 103.4 μM, 81.40 μM, 71.84 μM ( Figure 1 ).

[0035] 2.2 Detection of the antiviral ability of Alloimperatorin Passage HEK293 cells into 96-well culture plates and inoculate with BPIV3 (MOI = 0.1), SeV (100 HAU / mL), VSV (MOI = 0.1), HSV-1 (MOI = 0.1); passage BHK-21 cells into 96-well culture plates and inoculate with BPIV3 (MOI = 0.1), BEFV (MOI = 0.1); passage MDBK cells into 96-well culture plates and inoculate with BPIV3 (MOI = 0.1), BEFV (MOI = 0.1). Incubate in a 5% CO₂ cell culture incubator at 37 °C for 1.5 h, and then add cell maintenance medium containing 2% serum with different concentrations of Alloimperatorin (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 μg / mL). Continuously observe for 4 - 7 days and calculate the IC 50 and SI values of Alloimperatorin against the virus.

[0036] Among them, the SI values of Alloimperatorin against the virus are shown in Table 1.

[0037] Table 1

[0038] As Figure 2 shown, the IC 50 values of Alloimperatorin against BPIV3, SeV, VSV, and HSV-1 in HEK293 cells were 15.17 μM, 13.07 μM, 12.74 μM, and 12.39 μM, respectively; the IC 50 values of Alloimperatorin against BPIV3 and BEFV in BHK-21 cells were 13.75 μM and 12.61 μM, respectively; the IC 50They were 9.36 μM and 8.87 μM respectively, and the SI values were all greater than 1 (Table 1), indicating that Alloimperatorin has the potential to resist the above viruses.

[0039] 2.3 Alloimperatorin inhibits virus replication Subculture HEK293 cells in 6-well culture plates. After growing to confluence, inoculate BPIV3 (MOI = 0.1), SeV (100 HAU / mL), VSV (MOI = 0.1), and HSV-1 (MOI = 0.1); subculture BHK-21 cells in 6-well culture plates. After growing to confluence, inoculate BPIV3 (MOI = 0.1) and BEFV (MOI = 0.1) respectively; subculture MDBK cells in 6-well culture plates. After growing to confluence, inoculate BPIV3 (MOI = 0.1) and BEFV (MOI = 0.1) respectively. Incubate in a 5% CO2 cell culture incubator at 37 °C for 1.5 h, then add cell culture medium containing 2% serum with Alloimperatorin (50 μg / mL). Harvest the virus after 24 h and measure the TCID of the virus solution. 50 。

[0040] The results are as Figure 3 shown. After treatment with Alloimperatorin, the virus titers of BPIV3, SeV, VSV, HSV-1, and BEFV were significantly lower than those of the control group, indicating that Alloimperatorin inhibits the replication of BPIV3, SeV, VSV, HSV-1, and BEFV.

[0041] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of alloimperatorin in the preparation of antiviral products; wherein, The virus includes at least one of bovine parainfluenza virus type III, Sendai virus, vesicular stomatitis virus, bovine ephemeral fever virus, and herpes simplex virus type I.

2. The application according to claim 1, characterized in that, The antiviral activity is manifested as the ability to inactivate the virus and / or inhibit virus replication.

3. The application according to claim 1, wherein The product is a drug or a test reagent, and the test reagent is for basic research use.

4. The application according to claim 1, characterized in that, The application of Alloimperatorin as an active ingredient or one of the active ingredients in the preparation of an antiviral product; preferably, the concentration of Alloimperatorin is 50 μg / mL.

5. The application of Alloimperatorin in the preparation of a product for inactivating the virus.

6. The application of Alloimperatorin in the preparation of a product for inhibiting virus replication.

7. The application according to claim 5 or 6, characterized in that, The virus includes at least one of bovine parainfluenza virus type III, Sendai virus, vesicular stomatitis virus, bovine ephemeral fever virus, and herpes simplex virus type I.

8. A pharmaceutical composition, characterized in that, The drug is composed of Alloimperatorin and at least one other pharmaceutically active ingredient and / or at least one other non-pharmaceutically active ingredient.

9. The pharmaceutical composition according to claim 8, wherein The non-pharmaceutically active ingredient includes a pharmaceutically acceptable carrier, excipient, and / or diluent.

10. The pharmaceutical composition according to claim 8, wherein The dosage forms of the pharmaceutical composition include: liquid dosage forms, solid dosage forms, topical preparations, and sprays.

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