Coumarins and Hesperetin Inhibit Human Respiratory Syncytial Virus Infection

Figures

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  Figure 1. Cytotoxicity assay by MTT of natural coumarins on HEp-2 cells following 48 h of incubation with the test compounds coumarin, esculetin, esculin, 4-methyl esculetin, and umbelliferone. The hexagon with dotted red line represents Staurosporine (1 mM), negative control, in which 50% of the cells remain viable.

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  Figure 2. (A) Fluorescence microscopy images taken using the green fluorescent protein (GFP) filter at 24, 48, and 72 h to demonstrate the spread of virus infection in treated cells compared with the untreated control or the DMSO-treated control. Treatment of rgRSV infection of HEp-2 cells with coumarin, esculetin, esculin, 4-methyl esculetin, and umbelliferone (before and after infection). HEp-2 cells were cultured overnight and inoculated with rgRSV. (B) The black line represents RSV infection in the presence of the coumarins, the red line is the DMSO solvent control, and the blue line represents the infection without compounds.

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  Figure 3. Inhibition assay with RSV-luc on A549 cells at 48 h for coumarins and 24 h for hesperetin at. The black lines and circles represent RSV infection in the presence of the compounds (coumarin, esculetin, esculin, 4-methyl esculetin, umbelliferone and hesperetin), red lines and squares represent RSV infection in the presence of the DMSO, and blue lines and triangle represents RSV infection in the absence of a compound.

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  Figure 4. Inhibition plot of RSV-luc infection of A549 in the presence of compounds esculetin, 4-methyl esculetin, and hesperetin, at 24 and 48 h.

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  Figure 5. Inhibition assay on A549 cell with RSV-luc for acetylated and prenylated umbelliferone at 24 h. The black lines and circles represent RSV infection in the presence of the compounds (coumarin, esculetin, esculin, 4-methyl esculetin, umbelliferone and hesperetin), red lines and squares represent RSV infection in the presence of the DMSO (solvent), and blue lines and triangles represent RSV infection without DMSO or compounds.

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  Figure 6. Inhibition assay of the encapsulated compounds on differentiated HBE cultures inoculated with RSV-luc, at 48 h. The black line and circles represents RSV infection in the presence of the encapsulated compounds (coumarin, esculetin, esculin, 4-methyl esculetin, umbelliferone and hesperetin), the red line and squares shows the RSV infection in the presence only of β-cyclodextrin, and the blue line and triangle represents the RSV infection with no additions.

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  Figure 7. RSV replication and spread in different cell lines and in primary HBE cultures in the presence of coumarins, hesperetin and modified umbelliferones. GFP-expressing RSV replication and spread in (A) HEp-2 cells as measured by virus yield in focus forming units ± standard deviation (SD). Replication and spread of RSV expressing luciferase, measured in relative light units (RLU) ± SD in A549 cells (B,D), and (C) HBE cultures. Statistical significance Significant differences are indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 and ns is non- significant.