Application of lacquer extract in preparing drugs for treating or preventing Zika virus

Abstract

The present invention relates to the application of lacquer extract in the preparation of anti-Zika virus drugs for treatment or prevention, and belongs to the field of biomedicine technology. The present invention relates to the application of two compounds extracted from lacquer in anti-Zika virus. Through cytotoxicity experiments, the compounds are detected to inhibit virus replication and the expression of Zika virus E protein, confirming that the two plant monomers are low-toxic and high-antiviral activity compounds, and have the potential value of becoming anti-Zika virus drugs, providing a theoretical and experimental basis for finding new breakthroughs in clinical anti-Zika virus drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to the application of a lacquer extract, and specifically relates to the application of a lacquer extract in the preparation of a drug for treating or preventing Zika virus. Background Art

[0002] Zika virus belongs to the Flaviviridae family and is a typical positive-sense single-stranded RNA virus transmitted by mosquitoes. SZWIV01 is its subtype. The viral genome is about 11kb in length and encodes a single open reading frame (ORF) flanked by two untranslated regions (UTRs). ORF can translate a polyprotein, which is processed into three structural proteins and seven non-structural (NS) proteins under the joint action of host and viral proteases. The structural proteins are capsid protein (C), premembrane and membrane protein (prM / M) and envelope protein (E), and the non-structural proteins are NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The three structural proteins mainly play a role in the formation and binding of viral particles, the fusion of viral envelope and host cell membrane, and the entry into host cells. The seven non-structural proteins are mainly involved in viral replication, RNA release, and escape from the host immune response.

[0003] ZIKV infection is mainly transmitted from mosquito to human and from Aedes mosquitoes. Mosquitoes are the most important vector for transmitting the virus to humans, in addition to sexual transmission, blood transfusion and mother-to-child transmission. Symptoms of ZIKV infection may appear around 3-14 days after infection, and most people infected with ZIKV have no or only mild symptoms. Its common symptoms include headache, fever, myalgia, arthralgia, maculopapular rash and conjunctivitis. There is increasing evidence that Zika virus infection can lead to some serious neurological complications, such as Guillain-Barré syndrome and congenital Zika syndrome. So far, there is no protective vaccine to prevent infection and no antiviral drugs to treat infection.

[0004] Euphorbia helioscopia L. is called "zeqi" in Chinese. It belongs to the Euphorbiaceae family, Euphorbia genus, and is an annual herb. For decades, its aerial parts have been widely used as folk medicine in Sichuan, Guizhou, Jiangxi, Hunan and other places to treat various diseases, including edema, cough, malaria, bacillary dysentery, warts, tuberculous fistula, osteomyelitis and tumors. In recent years, zeqi has received increasing attention due to its diverse chemical components, wide range of biological properties and great medical value.

[0005] The present invention found that two compounds extracted from lacquer have the function of resisting Zika virus, and the chemical structural formula of the compounds is as follows:

[0006]

[0007] A search revealed no reports that these two compounds have anti-Zika virus properties. Summary of the invention

[0008] In view of this, the purpose of the present invention is to provide an application of an extract of Rhizoma Zosterae in the treatment of flavivirus, especially Zika virus infection, so as to fully explore the medical value of Rhizoma Zosterae and open up a new approach of Chinese herbal medicine for the treatment of Zika infection.

[0009] The solution of the present invention is achieved through the following technologies:

[0010] The present invention provides application of a lacquerwood extract in preparing a drug for treating or preventing Zika virus.

[0011] Furthermore, the structure of the lacquer extract is as follows:

[0012]

[0013] Preferably, the Zika virus is Zika virus SZWIV01.

[0014] Furthermore, the medicine contains the extract of Rhizoma Coptidis and pharmaceutically acceptable excipients.

[0015] The present invention has the following advantages: the application of the two compounds extracted from zelkova in the present invention in anti-Zika virus, through cytotoxicity experiments, detecting the compounds inhibiting virus replication and the expression of Zika virus E protein, confirming that the two plant monomers are low-toxic and high-antiviral activity compounds, and have the potential value of becoming anti-Zika virus drugs, providing a theoretical and experimental basis for finding a new breakthrough in clinical anti-Zika virus drugs.

[0016] Attached picture content:

[0017] Figure 1 This is a graph showing the toxicity of zeqi compounds to Vero cells;

[0018] Figure 2 The protective effect of the zeqi compound on ZIKV-infected Vero cells;

[0019] Figure 3Representative images and gray value statistics of immunoblot analysis of the inhibitory effect of zechaic compounds on Zika E protein in ZIKV-infected Vero cells. (ns, no statistical difference; **p<0.01, ***p<0.001, ****p<0.0001)

[0020] Figure 4 Immunofluorescence was used to detect the inhibitory effect of zechaic compounds on Zika E protein. (Blue nucleus; green E protein) DETAILED DESCRIPTION

[0021] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0022] Example 1: Extraction of lacquer compounds

[0023] The whole plant of Ze Lacquer (10 kg) was air-dried and then crushed. It was extracted with methanol for 5 times under heating in a water bath at 60°C, each time for 24 hours. The methanol extract was vacuum concentrated to obtain Ze Lacquer extract (1.5 kg). The extract was extracted with ethyl acetate for 5 times, and the ethyl acetate soluble extract (400 g) was obtained by vacuum concentration. The ethyl acetate extract (400 g) was treated with D-101 non-polar macroporous resin, and the methanol / water system (50%, 70%, and 90%) was used as the mobile phase for gradient elution to obtain the fraction Fr.C. It was further separated and purified, and the eight components Fr.C1-Fr.C8 were obtained by gradient elution on a (100-200 mesh) silica gel column with a petroleum ether / ethyl acetate (15:1, 10:1, 5:1, 3:1, 2:1, 1:1, EtOAc, MeOH) system. The Fr.C3 segment was first decolorized with MCI material, and gradient eluted with methanol / water (50%-100%) as the eluent, and then combined by thin layer color development to obtain six components Fr.C3a-Fr.C3f. The Fr.C3e component was gradient eluted with silica gel as the stationary phase and petroleum ether / ethyl acetate as the mobile phase (10:1, 7:1, 4:1), and three components (Fr.C3e1-Fr.C3e3) were obtained. Fr.C3e3 was initially separated and purified by preparative high performance liquid chromatography (MeCN / H2O, 70:30, v / v), and three fractions (Fr.C3e3a, Fr.C3e3b and Fr.C3e3c) were obtained. Fr.C3e3b was then separated and purified by preparative plate under petroleum ether / acetone (8:1) to obtain compound 2 (15.2 mg). Fr.C3d was first purified by elution on a Sephadex LH-20 gel column with pure methanol, and then further separated by semi-preparative high performance liquid chromatography (MeCN / H2O, 70:30, v / v) to obtain four components (Fr.C3d1-Fr.C3d4). Fr.C3d3 was eluted on a silica gel column with a gradient of chloroform / acetone (80:1), and the main component was eluted on a silica gel column with petroleum ether / acetone (7:1) to obtain compound 1 (73 mg).

[0024] Example 2 Structure of the compound and cytotoxicity test

[0025] The two lacquer compounds provided by the present invention belong to diterpenes in chemical structure, and their structural formulas and spectral data are as follows:

[0026]

[0027]

[0028] Experimental materials: cells and viruses

[0029] African green monkey kidney cells (Vero) were grown and maintained in Dulbecco's modified medium (DMEM, VivaCell, China) containing 10% fetal bovine serum (FBS, GIBCO, USA) and 1% gentamicin at 37°C, 5% CO2. C6 / 36 (Aedes albopictus) cells were used to amplify ZIKV, and the cells were cultured in Roswell Park Memorial Institute-1640 medium (RPMI-1640; VivaCell, China) containing 10% FBS. The ZIKV SZWIV01 strain (GenBank: KU963796) was obtained from the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0030] To determine whether the zeqi compound has an effect on cell activity, the cytotoxicity of the zeqi compound was detected by the CCK-8 method. Vero cells were plated in 96-well plates at 5000 cells / well and cultured in a 37°C, 5% CO2 incubator until they were completely attached. The zeqi compound was gradiently diluted with 2% FBS DMEM maintenance medium to 200μM, 100μM, 50μM, 25μM, 12.5μM, and 6.25μM and added to each well, with 2 replicates for each concentration. At the same time, a negative control group and a blank group without compounds were set up, and the cells were cultured in a 37°C, 5% CO2 incubator for 3 days. After 3 days, new culture medium was replaced, 20uL CCK-8 reagent was added to each well, and the OD value at a wavelength of 450nm was measured with an enzyme marker after incubation at 37°C for 2h. The OD values ​​under different concentration gradients were recorded, and the inhibition rate of the compound on cell viability was calculated by comparing with the blank control and positive control groups. Cell viability (Cell Viability%) = (OD value of experimental well - OD value of blank well) / (OD value of control well - OD value of blank well) × 100%

[0031] Cytotoxicity of lacquer compounds Figure 1 As shown, the results show that the EC of the two lacquer compounds 50 The values ​​were all >200 μM, indicating that they were less toxic to Vero cells. The cytotoxicity results showed that the two lacquer compounds were compounds with low cytotoxicity and were worthy of further study on their anti-ZIKV activity.

[0032] Example 3: Zest compounds inhibit Zika virus replication

[0033] To verify the antiviral activity of the zeqi compound, the inhibitory effect of the zeqi compound on progeny virus particles was detected by qRT-PCR. Vero cells were cultured at 3×10 5The cells were plated in 12-well plates and cultured overnight in a 37°C, 5% CO2 incubator. After the cells grew to a fully adherent state, ZIKV was added at an MOI of 0.1 to infect the cells for 4 hours. After the infection, the cells were washed three times with PBS to remove the unadsorbed viruses. Then different concentrations of lacquer compounds (1μM, 2.5μM, 5μM, 10μM, 25μM, 50μM) were added, and the supernatant was collected after continued culture for 72 hours. The supernatant RNA was extracted using the Viral DNA / RNA Kit (Beijing Quanshijin Biotechnology Co., Ltd.) according to the kit instructions, and then the ZIKV viral load was detected using the one-step quantitative kit Evo M-MLVOne Step RT-qPCR KitⅢ (Probe), ROX dye and TaqMan probe under the QuantStudio 5Real-Time PCR system.

[0034] The results are as follows Figure 2 As shown in Figure 2, both lacquer compounds exhibited good anti-ZIKV activity and inhibited the release of progeny virus particles in a dose-dependent manner. Their IC 50 The values ​​were 2.63 and 5.94 μM respectively. The experimental results showed that both compounds had good anti-ZIKV activity, could inhibit the release of progeny viruses, and had the potential value of becoming anti-ZIKV drugs.

[0035] Example 4: Anti-Zika virus activity detection of zeqi compounds

[0036] To further confirm the anti-Zika virus activity of the Zeqi compound, the inhibitory effect of the Zeqi compound on the E protein was detected by Western blot. Zika virus infected Vero cells at MOI=1 for 4 hours, and washed three times with PBS after the infection. Then different concentrations of compounds (20μM, 10μM, 5μM, 2.5μM and 1.25μM) and maintenance medium were added and cultured for 72 hours. After 72 hours, the cell supernatant was removed, the cells were washed 2-3 times with PBS, and then the proteins were collected with RIPA buffer supplemented with protease and phosphatase inhibitors and the cells were lysed on ice for 1 hour, followed by centrifugation at 4°C and 12000rpm / min for 10 minutes to collect the cell lysate. The protein concentration in the cell lysate was measured by the bicinchoninic acid (BCA) protein assay. Subsequently, the inhibitory effect of the compound on the Zika virus envelope protein (E-protein) was detected by Western blot, and the grayscale value statistical analysis was performed by ImageJ. The results are shown in Figure 2. Figure 3As shown, similar to the results of inhibiting viral replication, the compounds inhibited the expression of Zika virus E protein in a concentration-dependent manner. The two compounds almost completely inhibited Zika virus E protein at a concentration of 10 μM, which was better than the positive control drug 50 μM ribavirin (RBV). The results proved that both compounds have good anti-Zika virus activity.

[0037] Example 5: Zeqi compound significantly inhibited the expression of Zika virus E protein

[0038] To confirm the inhibitory effect of the zeqi compound on the intracellular Zika virus E protein, the compound inhibited the expression of Zika E protein by immunofluorescence detection. Vero cells were infected with Zika virus at MOI = 1 for 4 hours, and then cultured with different concentrations of compounds and maintenance medium for 72 hours. After the culture, the supernatant was removed, the cells were washed 2-3 times with PBS, and then fixed with 4% paraformaldehyde for 30 minutes, and permeabilized with PBS containing 0.1% TritonX-100 for 15 minutes. After washing with PBS, the cells were blocked with PBS blocking solution containing 5% BSA for 1 hour. Then incubated with ZIKV E antibody at 4°C overnight, and incubated with goat anti-rabbit IgG h&L (Alexa Fluor) the next day. 488) secondary antibody (Abcam) was incubated for 1 hour. The cells were stained with 40,6-diamidino-2-phenylindole dihydrochloride (DAPI) and photographed using a Motic fluorescence microscope. Figure 4 As shown: (green ZIKVE protein, blue nucleus) Corresponding to the Western blot test results, the two compounds significantly inhibited the expression of Zika virus E protein in Vero cells compared with the negative control group without compound addition. Only a very small amount of E protein expression was detected in Vero cells treated with 10μM compound, which was better than the positive control group treated with 50μM ribavirin.

Claims

1. Use of compound 1 or 2 extracted from lacquer in the preparation of a drug for treating or preventing Zika virus, characterized in that: The structure of the compound 1 or 2 extracted from the lacquer is shown below: .

2. The use according to claim 1, characterized in that: The Zika virus is Zika virus SZWIV01.

3. The use according to claim 1 or 2, characterized in that: The medicine contains compound 1 or 2 extracted from lacquer and pharmaceutically acceptable excipients.

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