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. 2022 Apr 25;15(5):530. doi: 10.3390/ph15050530

Figure 6.

Figure 6

Hypericum perforatum (HP1) and hypericin (HY) carry direct SARS-CoV-2 virus-blocking activities. (A–C) Vero cells were seeded overnight. The next day, different treatment protocols with HP1 or hypericin were applied. The treatment conditions included (i) only pre-treatment of cells (for 1 h at 37 °C) in infection-DMEM containing solvent control (DMSO) or HP1 or hypericin, (ii) only pre-treatment of SARS-CoV-2 (for 1 h at room temperature) in infection-PBS containing solvent control or HP1 or hypericin, or (iii) only post-treatment of cells after infection in infection-DMEM (at 37 °C) containing solvent control or HP1 or hypericin. As control, the combined treatment protocol of pre-treatment of cells and SARS-CoV-2 and post-treatment of cells was included as well (Full treatment). The SARS-CoV-2 infection was conducted at MOI of 0.05 or 1, as the total length of the infection experiment was (A,B) 24 h or (C) 8 h, respectively. (A–C) After the depicted length of experiments, virus supernatants were harvested, virus titration was done with plaque assay, results are shown as PFU/mL (means and s.d.), and two-way ANOVA with Sidak’s multiple comparisons was done by comparing each value to its respective solvent control. (D) SARS-CoV-2 was incubated for 1 h at room temperature with solvent control (DMSO) or HP1 or hypericin in an infection-PBS mix and directly submitted to plaque assay. Obtained data are expressed as PFU/mL (mean and s.d.), and one-way ANOVA with Dunnett’s multiple comparisons was done by comparing each value to the solvent control. (E) After Vero cells were seeded on cover slips overnight, cells were infected with 1 h pre-treated (either with DMSO or 15 µg/mL HP1 or 100 ng/mL hypericin (HY)) SARS-CoV-2 virus. Mock-infected cells served as control. Then, 2, 4, 6, and 8 h after infection, cold methanol (–20 °c) was used for cell fixation, and indirect immunofluorescence staining of the SARS-CoV-2 nucleoprotein (green) and nuclei (blue) was conducted. Exposure times for each channel where fixed on the 8 h infected and DMSO-treated samples (scale bar represents 50µM). (F) The day after seeding, Vero cells were infected with SARS-CoV-2 (MOI = 0.05) for 1 h. After infection, cells were either incubated with solvent control or hypericin (250 ng/mL) in infection-DMEM. Supernatants were collected 24 h after infection and submitted to plaque assay. Obtained data are shown as PFU/mL, and Student’s t-test with Welch’s corrections was done. n.d means non-detected. * for p ≤ 0.05, ** for p ≤ 0.01, and **** for p ≤ 0.0001.