Application of angelica lactone A in the preparation of antiviral preparations against filovirus

Abstract

The present invention relates to the technical field of novel drug applications, and more specifically, to the use of angelica lactone A in the preparation of antiviral preparations for filoviruses. Angelica lactone A inhibits the replication of filoviruses, such as Ebola and Marburg viruses, providing a safe and effective new drug option for the clinical treatment of Ebola and other filovirus infections.

Description

Technical Field

[0001] The present invention relates to the technical field of new drug applications, and in particular to the application of angelica lactone A in the preparation of anti-filamentous virus antiviral preparations. Background Art

[0002] Filovirus is an enveloped, single-stranded, non-segmented RNA virus belonging to the order Single-stranded negative-strand RNA Virales. It is a virus that can infect vertebrates. The Filoviridae family includes the genus Ebolavirus, the genus Marburgvirus, and the genus Cuevavirus. Among them, the genus Ebolavirus includes six viruses, namely Ebola virus (EBOV), Sudan virus (SUDV), Tai Forest virus ( Forest virus (TAFV), Bundibugyo virus (BDBV), Reston virus (RESTV) and Bombali virus (BOMV); the Marburgvirus genus includes Marburg virus (MARV); the Quevavirus genus includes Lloviu virus (LLOV).

[0003] Ebola virus (EBOV) infection in humans can cause a severe, often fatal, hemorrhagic, and febrile illness (with a mortality rate of up to 90%), hence the name Ebola hemorrhagic fever (EHF), also known as Ebola virus disease (EVD). According to the World Health Organization, since EBOV was first discovered in 1976, EVD outbreaks have occurred over 40 times. The 2014-2016 West African EVD outbreak alone reported 28,000 confirmed cases (with a mortality rate exceeding 40%), with cases exported to the United States and Europe. In recent years, due to the increasing frequency of EVD outbreaks and cross-border transmission, filoviruses such as EBOV have garnered widespread attention from scholars and the public both domestically and internationally, earning them the nickname "the black ghost lurking in the African jungle." Furthermore, reports indicate that filoviruses are also present in my country, posing a potential threat of outbreaks. Therefore, there is an urgent need to develop effective treatments for EBOV infection to better address outbreaks.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to provide an application of angelica lactone A. The present invention provides a new application of angelica lactone A, which inhibits the replication of filoviruses such as Ebola virus and Marburg virus, providing a safe and effective new drug option for the clinical prevention and treatment of filovirus infections such as Ebola virus.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides the use of angelica lactone A in the preparation of an antiviral preparation against filovirus.

[0008] In alternative embodiments, the filovirus includes Ebola virus and Marburg virus.

[0009] In an alternative embodiment, the filovirus comprises Bola virus, Sudan virus, Tai Forest virus, Bundibugyo virus and Marburg virus. In an alternative embodiment, the antiviral agent is a drug that inhibits viral replication.

[0010] In a second aspect, the present invention provides the use of angelica lactone A in the preparation of a medicament for treating diseases caused by filovirus infection.

[0011] In alternative embodiments, the disease comprises Ebola hemorrhagic fever and Marburg hemorrhagic fever.

[0012] In a third aspect, the present invention provides the use of angelica lactone A as the sole active ingredient in the preparation of a drug for treating diseases caused by filovirus infection.

[0013] In a fourth aspect, the present invention provides the use of angelica lactone A in the preparation of an antiviral preparation for Ebola virus.

[0014] In a fifth aspect, the present invention provides the use of angelica lactone A in the preparation of a medicament for treating diseases caused by Ebola virus infection.

[0015] In a sixth aspect, the present invention provides the use of angelica lactone A in the preparation of an antiviral preparation against Marburg virus.

[0016] The present invention has the following beneficial effects: Angelica lactone A has a good anti-infection effect on members of the Ebola virus genus, such as SUDV, TAFV and BDBV, and also has a good inhibitory effect on the replication of MARV, indicating that it can effectively inhibit filovirus infection and can be used as a drug for treating filovirus infections such as EBOV, which further expands the application scope of Angelica lactone A. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a test result diagram provided for Experimental Example 1 of the present invention;

[0019] Figure 2 This is a test result diagram provided for Experimental Example 2 of the present invention;

[0020] Figure 3 This is a test result diagram provided for Experimental Example 3 of the present invention;

[0021] Figure 4 This is a test result diagram provided for Experimental Example 4 of the present invention;

[0022] Figure 5 This is the test result diagram provided for Experimental Example 5 of the present invention. DETAILED DESCRIPTION

[0023] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0024] The structural formula of Levistolide A (LA) is as follows:

[0025] It is a phthalide dimer and one of the dimer products of ligustilide, the main active ingredient of the volatile oils of Chuanxiong and Angelica. Ligustilide A belongs to the type II structure of ligustilide. Ligustilide A has multiple pharmacological properties, including anti-inflammatory and anti-cancer activities, and can regulate the ROS pathway to exert antiviral effects. In addition, ligustilide A can also induce cell apoptosis and autophagy; and at the cellular level, ligustilide A shows significant activity in anti-liver cancer and other aspects, but there is no record or suggestion in the existing technology that ligustilide A has a good anti-infection effect against filamentous viruses such as EBOV and MARV.

[0026] The EBOV genome is approximately 19 kb long and contains seven gene segments, encoding seven structural proteins from the 3' to 5' ends: NP, VP35, VP40, GP, VP30, VP24, and L. Furthermore, during transcription, the GP gene undergoes RNA editing, resulting in frameshift translation, which produces two additional non-structural proteins (sGP and ssGP). To date, no small molecule drugs for EBOV and other filovirus infections have been approved by the FDA.

[0027] The inventors found that within the non-toxic range, angelica lactone A not only has a good inhibitory effect on EBOV, but also has a good anti-infection effect on SUDV, TAFV and BDBV of the Ebola virus genus, and also has a significant inhibitory effect on the replication of MARV, indicating that angelica lactone A can be used as an anti-filamentous virus infection preparation.

[0028] It should be noted that the angelica lactone A used in the examples of the present invention is a purchased raw material, for example, from raw material manufacturers such as Chengdu Desite Biotechnology Co., Ltd., Chengdu Geput Biotechnology Co., Ltd., Shanghai Xige Biotechnology Co., Ltd., and Chengdu Pufeide Reference Material Technology Co., Ltd. Angelica lactone A can also be prepared by referring to existing literature.

[0029] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0030] Experimental Example 1

[0031] Drug inhibition of EBOV minigenome replication experiment

[0032] method:

[0033] The EBOV minigenome system was transfected into a 96-well plate, and 100 μM, 50 μM, 25 μM, 12.25 μM, 6.125 μM, 3.0625 μM, 1.53125 μM, and 0.7656 μM of angelica lactone A (diluted in DMEM containing 2% FBS and 1% double antibody) were added 6 h after transfection. At the same time, a group with the same amount of DMSO was set as a control. After incubation in a 37°C incubator for 48 h, the supernatant was discarded, the cells were collected and lysed, and the luciferase activity was read to obtain the replication of the EBOV minigenome after treatment with different concentrations of angelica lactone A.

[0034] Results see Figure 1 ,according to Figure 1 It can be seen that angelica lactone A can effectively inhibit the replication of EBOV mini-genome in cells, IC 50 It is 14.72μM.

[0035] Experimental Example 2

[0036] Drug cytotoxicity experiments

[0037] method:

[0038] The CCK-8 kit is used to measure drug cytotoxicity. WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylphenyl)-2H-tetrazolium monosodium salt) in the kit is reduced to a highly water-soluble orange-yellow formazan by mitochondrial dehydrogenases under the action of the electron-coupled carrier 1-methoxy PMS. The color of the resulting formazan is directly proportional to cell viability and inversely proportional to cytotoxicity. After incubating cells with varying drug concentrations for the appropriate time, CCK-8 is added and absorbance is measured at 450 nm using a microplate reader. This value indirectly reflects the number of viable cells.

[0039] Results see Figure 2 ,according to Figure 2 It can be seen that the set drug concentrations are 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM and 0.78125 μM, respectively.

[0040] Experimental Example 3

[0041] Drug inhibition of EBOV infection experiment

[0042] method:

[0043] HeLa, Huh-7 and other cell lines were used as viral infection models. HeLa cells were infected with MOI=1 and Huh-7 cells were infected with MOI=0.1. One hour after infection, the culture medium was aspirated and discarded. After washing the cells twice with PBS buffer, 10μM, 1μM, 0.1μM, 0.01μM and 0.001μM of angelica lactone A (diluted with DMEM containing 2% FBS and 1% double antibody) were added respectively. At the same time, a control group without drug addition was set up and cultured in a 37°C incubator. After 72 hours, the cell supernatant was collected, and the viral RNA in the supernatant was extracted using a kit. The viral content in the supernatant after treatment with different drugs was detected by RT-qPCR, and the drug inhibition rate was calculated.

[0044] Results see Figure 3 ,according to Figure 3 It can be seen that angelica lactone A can effectively inhibit EBOV infection in HeLa and Huh-7 cells, IC 50 0.0510 μM and 0.2495 μM respectively.

[0045] Experimental Example 4

[0046] Experiment on drug inhibition of different filovirus infection

[0047] method:

[0048] The Huh-7 cell line was used as a viral infection model. Huh-7 was infected with SUDV, BDBV and TAFV (MOI = 0.1), respectively. One hour after infection, the culture medium was aspirated. After washing the cells twice with PBS buffer, 10 μM, 1 μM, 0.1 μM, 0.01 μM and 0.001 μM of angelica lactone A (diluted with DMEM containing 2% FBS and 1% double antibody) were added, respectively. At the same time, a control group without drug addition was set up and the cells were placed in a 37°C incubator for continued culture. After 72 hours, the cell supernatant was collected, and the viral RNA in the supernatant was extracted using a kit. The viral content in the supernatant after treatment with different drugs was detected by RT-qPCR, and the drug inhibition rate was calculated.

[0049] Results see Figure 4 ,according to Figure 4 It can be seen that angelica lactone A can effectively inhibit the infection of Ebolaviruses such as SUDV, BDBV and TAFV in Huh-7 cells.

[0050] Experimental Example 5

[0051] Drug inhibition of MARV minigenome replication experiment

[0052] method:

[0053] The MARV minigenome system was transfected into a 96-well plate, and 200 μM, 40 μM, 8 μM, 1.6 μM, 0.32 μM, and 0.064 μM of angelica lactone A (diluted in DMEM containing 2% FBS and 1% double antibody) were added 6 h after transfection. At the same time, a group with the same amount of DMSO was set as a control. After continued culture in a 37°C incubator for 48 h, the supernatant was discarded, the cells were collected and lysed, and the luciferase activity was read to obtain the replication of the MARV minigenome after treatment with different concentrations of angelica lactone A.

[0054] Results see Figure 5 ,according to Figure 5 It can be seen that angelica lactone A can effectively inhibit the replication of MARV minigenome in cells.

[0055] Combined with the previous analysis, angelica lactone A can effectively inhibit the infection of Ebola virus and filovirus such as Marburg virus.

[0056] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of angelica lactone A in the preparation of an antiviral preparation against filovirus, wherein the filovirus is a virus of the genus Ebola.

2. The use according to claim 1, characterized in that The Ebola virus is selected from the group consisting of Bola virus, Sudan virus, Tai Forest virus and Bundibugyo virus.

3. The use according to claim 1, characterized in that The antiviral preparation is a drug that inhibits viral replication.

4. The use according to claim 1, characterized in that The application also includes application in preparing medicines for treating diseases caused by Ebola virus infection.

5. The use according to claim 4, characterized in that Such diseases include Ebola and Marburg hemorrhagic fevers.

6. Application of angelica lactone A in the preparation of antiviral preparations against Marburg virus.

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