Oro-nasal film spray with andrographolide blocks SARS-CoV-2 and influenza infections and strengthens mucosal innate immunity

Background: Currently, there are no specific treatments for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza and respiratory syncytial virus (RSV) infection. While phytochemicals have been explored for preventing viral entry, few approaches simultaneously target viral binding and enhance mucosal innate immunity. This study aimed to develop an oro-nasal film spray containing andrographolide and evaluate its antiviral, mucosal immune-enhancing, and anti-inflammatory effects. Methods: The antiviral activity of andrographolide and the formulated spray was assessed using a SARS-CoV-2 spike pseudovirus assay in HEK293-hACE2 cells expressing TMPRSS2. Plaque reduction assays were conducted against SARS-CoV-2, influenza A/H3N2, A/H1N1, and RSV. ELISA was used to measure mucosal immune markers (LL-37 and HD-5), while inflammatory cytokine levels were analyzed via multiplex bead immunoassay. Nitric oxide inhibition was used to assess anti-inflammatory activity. Results: The andrographolide spray (10 µg/ml) inhibited SARS-CoV-2 pseudovirus infection with ∼80% cell viability. It showed strong plaque reduction against SARS-CoV-2, partial inhibition of influenza A/H3N2, and no effect on RSV or A/H1N1. The spray significantly induced LL-37 and HD-5 expression in oral keratinocytes at 24 and 48 h and demonstrated dose-dependent anti-inflammatory effects. Conclusion: The andrographolide film spray exhibits dual functionality—antiviral activity against SARS-CoV-2 and influenza A/H3N2, and enhancement of mucosal innate immunity in vitro. These findings support its potential as a preventative strategy at the primary site of viral entry. Further in vivo investigation is warranted to explore and confirm its effects.