Evaluation of antiviral <i>in vitro</i> and <i>in silico</i> activity of <i>Psidium guajava</i> L alcoholic extract against Influenza A/(H1N1)pdm09

The emergence of extreme drug-resistant influenza strains necessitates the development of novel antiviral agents. Natural products offer a promising avenue for discovering effective treatments against influenza viruses. This study evaluated the antiviral potential of ethanolic and methanolic extracts of <i>Psidium guajava</i> L. against Influenza A/(H1N1)pdm09 virus using <i>in</i> <i>vitro</i> and <i>in silico</i> approaches. Cytotoxicity and antiviral activity were assessed in MDCK cells (CC₅₀ &gt; 100 µg/mL). Phytochemical analyses using FTIR and GC-MS were performed for the identification of major bioactive compounds and further evaluated for assessed antioxidant and anti-inflammatory properties. Molecular docking and dynamics simulation studies have explored potential mechanisms of action. The extracts exhibited significant antiviral effects, particularly in co-treatment, with ethanolic extract reducing viral load by 99.99% and methanolic extract by 99.96%. Post-treatment efficacy was lower but still notable. The key bioactive compounds, including γ-sitosterol and Caryophylla-4(12),8(13)-dien-5α-ol, demonstrated strong binding affinities to viral proteins such as hemagglutinin, M2, neuraminidase, and polymerase. The findings suggest that <i>P.</i> <i>guajava</i> extracts possess multi-targets antiviral activity against Influenza A virus with strong safety and therapeutic potential. The current study highlights the need for further pre-clinical and clinical evaluations to validate their efficacy as alternative antiviral agents. <b><i>Psidium guajava</i></b> Leaf extracts Show Promise Antiviral against Influenza A Virus: Influenza viruses continue to pose significant public health challenges worldwide, especially as some strains develop resistance to existing medications. Our research investigated whether extracts from <i>Psidium guajava</i> L leaves could effectively fight against the Influenza A/(H1N1)pdm09 virus (commonly known as “swine flu”). We tested two different <i>Psidium guajava</i> leaf extracts (prepared using ethanol and methanol as solvents) against Influenza A/(H1N1)pdm09 virus in laboratory experiments. We also used Molecular docking and simulation to understand how compounds from these extracts might interact with viral proteins. Additionally, we analyzed chemical composition of the extracts to identify the active ingredients. Both extracts showed strong antiviral effects against A/(H1N1)pdm09, especially when administered at the same time as virus exposure. Even when given after virus infection, extracts still showed beneficial effects. Importantly, extracts were safe for cells at the concentrations used. Chemical analysis revealed several natural compounds in the extracts, including γ-sitosterol and Caryophylla-4(12),8(13)-dien-5α-ol, which our computer modeling could bind to multiple viral proteins that are essential for viral infection and replication. Our findings suggest that <i>Psidium guajava</i> leaf extracts could be developed into alternative treatments for Influenza A virus infections. These natural extracts appear to work by targeting multiple viral proteins simultaneously, which might help prevent the development of drug resistance. Additionally, these extracts demonstrated antioxidant and anti-inflammatory properties, which could provide additional benefits during viral infections. Further research, including animal studies and clinical trials that may be required to confirm whether guava leaf extracts could become effective treatments for Influenza in humans.