Broccoli (Brassica oleracea L. var. Italica) leaves are a byproduct of broccoli and could be used as a food source. The study aimed to evaluate the effect of broccoli leaves on influenza A virus (IAV) infection. We investigated the effect of ethanol extract of Broccoli leaves (EBL) on IAV infection using green fluorescent protein (GFP)–tagged Influenza A/PR/8/34 virus (PR8-GFP IAV). When EBL and PR8-GFP IAV were cotreated to RAW 264.7 cells, the fluorescence microscopy and fluorescence-activated cell sorting (FACS) analysis showed that EBL significantly reduced the levels of GFP expression by influenza viral infection dose-dependently. Immunofluorescence (IF) analysis confirmed that EBL decreased the expression of IAV proteins. EBL exhibited a strong inhibitory effect of IAV binding on the cells and moderate virucidal impact. Consistently, EBL potently suppressed the hemagglutination by IAV infection. These results indicate that EBL prevents IAV attachment via the inhibition of HA upon viral infection. Finally, EBL as an HA inhibitor of IAV could be used as the natural antiviral source to protect against influenza viral infection.

Background: Morbidity and mortality as a result of influenza is an ongoing global health issue. Macrophages are part of the immune defense mechanism, as they play a role in the inflammatory response that blocks viral replication. Thuja orientalis Folium (TOF) has been prescribed traditionally as an expectorant for inflammatory airway disease. In this investigation, we have assessed the anti-viral properties of TOF water extract on the infectivity of the influenza A (H1N1) virus in RAW 264.7 macrophages.Method: We assessed the ability of GFP-tagged influenza A virus (A/PR/8/34-GFP) to infect murine macrophages. To determine the potential of antiviral activity using Fluorescence-activated cell sorting (FACS), fluorescent microscopy with green fluorescent protein (GFP)-tagged Influenza A/PR/8/34 virus. Hemagglutination inhibition assay, Western blotting, and Immunofluorescence analyses were performed to find the target protein. To investigate which stage of viral infection is associated with the TOF antiviral impact, we evaluated viral infection at three stages: attachment, entry, and virucidal in the presence of TOF.Results: FACS and immunofluorescence analyses results showed that TOF significantly inhibited viral infection and 100 μg/mL of TOF repressed that up to 98% of untreated control. Consistently, Western blot and immunofluorescence analyses against the influenza viral protein confirmed the TOF extract reduced the influenza viral protein expression in a dose-dependent manner. Time of addition and hemagglutination assays elucidated that TOF suppresses influenza A viral infection at early stages and interfere with HA protein.Conclusion: The TOF extract was thus found to exhibit inhibitory effects on influenza virus infection by inhibiting hemagglutination, blocking viral attachment and entry, and through virucidal effects. Based on our results, TOF may be a potential natural resource that could be utilized to develop an antiviral reagent.