In vitro and Computational Studies on the Anti-H1N1 Activity of Processed Citrus reticulata Peels

Abstract

Influenza A (H1N1) remains a persistent threat due to its rapid genetic mutation and resistance to antiviral drugs like Tamiflu. Citrus reticulata peels (chen-pi) are traditionally used in medicine and contain flavonoids with potential antiviral properties. However, the impact of different processing methods on its antiviral activity is not well understood. This study aims to evaluate the anti-influenza activity of processed Citrus reticulata peels using in vitro assays and computational methods, including network pharmacology and molecular docking, and to identify key bioactive compounds and target interactions. Three processing methods (soil, bran, and vinegar) were applied to Citrus reticulata peels, and their ethanol and water extracts were tested for anti-influenza activity using an MDCK cell model. Network pharmacology analysis identified potential target genes, and molecular docking assessed the binding affinity of hesperidin and narirutin to H1N1 nucleoprotein and key host proteins. Soil-processed ethanol extracts showed the highest anti-influenza activity (56.60 +/- 5.91%), followed by bran-processed water extracts (40.49 +/- 7.48%). Network pharmacology identified 11 target genes associated with the antiviral effect, with three key targets, including DOT1L, F2, and MMP2, playing significant roles. Molecular docking revealed that hesperidin and narirutin had higher binding affinities to DOT1L, F2, MMP2, and H1N1 nucleoprotein than Tamiflu, suggesting their potential as effective H1N1 treatments. The soil-processed ethanol extracts demonstrated notably superior anti-influenza activity, potentially due to enhanced interactions between bioactive compounds and soil-derived nutrients, thereby increasing pharmacological efficacy. Network pharmacology identified DOT1L, F2, and MMP2 as key target genes linked to antiviral activity, suggesting complex antiviral mechanisms involving host-pathogen interactions. Molecular docking analysis confirmed that hesperidin and narirutin had stronger binding affinities to these targets and H1N1 nucleoprotein compared to Tamiflu. These findings support Citrus reticulata peel extracts, particularly soil-processed ethanol extracts, as promising candidates for antiviral therapeutics and traditional medicinal applications, warranting further exploration for clinical use and development as alternative antiviral agents. This study highlights the importance of processing methods in optimizing the antiviral properties of Citrus reticulata peels. The findings support its application in traditional medicine and antiviral drug development, particularly as a potential alternative to existing antiviral treatments.