Anti-novel coronavirus drug and application thereof
A new type of coronavirus technology, applied in antiviral agents, drug combinations, antipyretics, etc., can solve the problem that there is no specific treatment for the disease
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Embodiment 1
[0020] This application selects VERO cells (African green monkey kidney cells) susceptible to the new coronavirus (2019-nCoV) as the research object (Vero is currently the main cell line used in in vitro experiments for drug screening of new coronaviruses). Culture conditions: DMEM containing 10% serum, placed at 37°C, containing 5% CO 2 cultivated in an incubator.
[0021] The new coronavirus used in the experiment was isolated from throat swabs of patients in Shenzhen Third People's Hospital in February 2020, and the sequence information has been uploaded to the GISAID database (BetaCoV / Shenzhen / SZTH-003 / 2020strainvirusGISAID access number: EPI_ISL_406594).
[0022] The resveratrol used in the experiment was purchased from SIGMA Company, product number (R5010, purity ≥ 99%), resveratrol was diluted to 0.1M with DMSO, and then diluted with DMEM containing 2% serum to ( 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM), for later culture.
[0023] The Vero ce...
Embodiment 2
[0025] In order to clarify the effect of resveratrol on cytotoxicity, this study was detected by CCK-8 kit. The CCK-8 reagent used in this experiment was purchased from China Suo Lai Bao Company (product number: CK04).
[0026] Vero cells were plated in 96-well plates after 4-5 passages, with 100 μl of cell suspension per well. The culture plate was pre-cultured in the incubator for 24 hours until the cells grew to 80%, the resveratrol was diluted to 0.1M with DMSO, and then the resveratrol was diluted with DMEM containing 10% serum to (1.56 μM, 3.13 μM , 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM), DMEM containing 0.05% DMSO was prepared with DMEM containing 10% serum as a drug blank control, and three wells were selected with only basal medium and CCK-8 reagent without The blank group of cell suspension was used as blank control. Add 10 μl of the above substances to be tested at different concentrations to the culture plate, each group has 3 repetitions, incubate in th...
Embodiment 3
[0034] This study needs to further clarify at what stage of viral infection resveratrol can play a role in order to further determine the main mechanism by which resveratrol may play a role.
[0035] The addition of resveratrol is mainly divided into four stages:
[0036] 1. Pre-infection treatment: Dilute resveratrol to 50 μM with DMEM containing 10% serum, pretreat the cells for 2 hours, then discard the pretreatment solution, wash three times with DMEM without serum, wash with DMEM without serum Dilute the virus, add the virus at MOI-0.01 and incubate for 2 hours, then wash the virus away and wash three times with serum-free DMEM. Subsequently, the cells were cultured with DMEM containing 2% serum until the sample was collected for 48 hours;
[0037] 2. Treatment during infection: Dilute resveratrol to 50 μM with serum-free DMEM to make solution A, then dilute the virus with solution A to MOI-0.01, incubate the cells for 2 hours, discard the incubation solution, and use se...
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