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Fig. 1 | Virology Journal

Fig. 1

From: Evaluation of the virucidal effects of rosmarinic acid against enterovirus 71 infection via in vitro and in vivo study

Fig. 1

Analysis of the virucidal effects of rosmarinic acid (RA) on EV71 infection. a RD cells (6 × 104) were infected with EV71 at multiplicity of infections (MOIs) of 0.1, 0.5, 1, and 3 and treated simultaneously with 1, 3, 5, or 10 μM RA for 24 h. Cell viability was determined by MTT assay. Percentage viability was calculated as (OD(infected + treated)–ODinfected)/ (ODinfected) × 100. b RD cells (6 × 104) were infected with EV71 virus at a MOI of 1 and treated simultaneously with or without RA. At 24 h post infection, the cytopathic effect was observed under a phase-contrast microscope (100× magnification). c Western blot analysis. RD cells (6 × 104) were infected with EV71and treated simultaneously with or without RA for 24 h at 37 °C. Cells were collected and the intracellular viral protein VP1 was detected by western blotting. β-actin was used as the loading control. d VP1 levels relative to β-actin were calculated. Cells treated with EV71 virus only were used as the positive control (100% reference). e Median cell culture infectious dose (CCID50). EV71 (MOI = 1; 6 × 104 PFU) was mixed with 0, 1, 3, 5, or 10 μM RA for 24 h at 37 °C. Supernatants were immediately added to RD cells to obtain the CCID50. The viral titer is presented as CCID50/mL (log10 EV71 titer). f Effects of RA on EV71-induced apoptosis. EV71-induced apoptosis was detected by flow cytometry following annexin V-FITC/PI double staining, and analyzed with Cell Quest software. Scatter diagram: Q2 = late apoptosis; Q3 = early apoptosis. g Total apoptosis (Q2 + Q3) is shown. Triplicate wells were used for each treatment. All results were obtained from three separate experiments and are presented as means ± S.D. h Time-dependent efficacy of RA against EV71 infection. Cells (6 × 104) were collected 24, 48, and 72 h post infection. Viability was assessed by MTT assay with blank cells (in DMEM with 10% FBS alone) set to 100%. In this study, blank control was uninfected-EV71 and no compounds in the DMEM-10%FBS media. Vehicle control was 0.5% PBS in DMEM-10%FBS media. Mock-treated control was treated-10 μM of RA in DMEM-10%FBS media. Infected control was infected-EV71 alone in DMEM-10%FBS media. Data were obtained from three separate experiments and are presented as the mean ± S.D. *P < 0.05, **P < 0.001, ***P < 0.0001, as compared with control

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