Anthos zenyth 200rt

Determination of the inhibitory concentration 50 (IC₅₀) was carried out as previously described by Mostafa, et al. [61 (link)]. In 96-well tissue culture plates, 2.4 × 10⁴ Vero-E6 cells were allocated in each well and incubated overnight at a humidified 37 °C incubator under 5% CO₂ condition. The cell monolayers were then washed once with 1× PBS and exposed to virus adsorption (hCoV-19/Egypt/NRC-03/2020 (Accession Number on GSAID: EPI_ISL_430820)) for 1 h at room temperature (RT). The cell monolayers were further overlaid with 100 μL of DMEM containing different serial concentrations of A. robusta bark EO as specified above in working solution. After incubation at 37 °C in 5% CO₂ incubator for 72 h, the cells were fixed with 100 μL of 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet in distilled water for 15 min at RT. The crystal violet dye was then dissolved using 100 μL absolute methanol per well and the optical density of the color is measured at 570 nm using Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The IC₅₀ of the sample is that required to reduce the virus-induced cytopathic effect (CPE) by 50%, relative to the virus control.

The IC₅₀ values for the tested compound were determined as previously described^{8 (link)}, with minor modifications. Briefly, the VERO-E6 monolayers in 96-well tissue culture plates were then washed once with 1 × PBS. The NRC-03-nhCoV” virus “TCID₅₀ = 100) was co-incubated with serial diluted working concentrations of the tested drugs at 37 °C for 1 h. The Vero-E6 cells were treated with virus/drug mixtures and kept at 37 °C for 1 h. Untreated/infected cells represented the virus control, however untreated/uninfected cells referred to the cell control. After 72 h of co-incubation at 37 °C in 5% CO₂ incubator, the cell monolayers were fixed with 100 μL of 10% formaldehyde for 20 min and stained with 0.1% crystal violet “in distilled water” for 15 min at RT. To dissolve crystal violet dye, 100 μL of the absolute methanol were added per well and the optical density of the color is eventually measured at 570 nm using the Anthos Zenyth 200rt plate reader (Anthos Labtec Instruments, Heerhugowaard, Netherlands). The IC₅₀ values were calculated using nonlinear regression analysis by plotting log inhibitor versus normalized response.