Abstract
Isatidis Radix, the root of Isatis indigotica, is a traditional Chinese medicine used against influenza. However, its chemical profile responsible for direct anti-influenza activity remains poorly characterized. This present study aims to classify the anti-influenza constituents of Isatidis Radix and to investigate their direct interactions with the key viral targets, neuraminidase (NA) and hemagglutinin (HA). To this end, a compound library comprising 91 constituents was assembled (78 isolated and 13 acquired), including nine undescribed compounds. Surface plasmon resonance (SPR) was employed to assess binding affinities to NA and HA. Antiviral activity of the compound library was validated in influenza A (H1N1)-infected A549 cells, and molecular docking simulations were performed to explore binding modes. SPR screening revealed that 63.7% and 71.4% of the library showed moderate-to-strong binding to NA and HA, respectively, with alkaloids and lignans as the predominant active classes. Notably, compounds 8, 15, 48, and 87 exhibited nanomolar KD values (0.49-4.22 nM for NA; 0.63-6.26 nM for HA), comparable to oseltamivir and arbidol. Cellular assays demonstrated that 76.9% of tested compounds showed antiviral activity (EC50 < 200 μM), with indole alkaloids and glycosylated lignans being most potent. Integrative SPR and cellular data confirmed a positive correlation between dual-target binding and antiviral efficacy. These findings establish that the anti-influenza activity of Isatidis Radix arises from multi-component, multi-target actions on NA and HA, providing a scientific basis for its clinical use and quality control.