High-Resolution Neuraminidase Inhibition Profiling of Synsepalum Dulcificum Based on UPLC-MS/MS: A Study of Anti-Influenza Constituents in a Potential Edible Resource.

Synsepalum dulcificum, commonly known as the miracle fruit, is a tropical plant noted for its unique biological activities. In this study, the anti-influenza constituents from S. dulcificum leaves were identified via neuraminidase (NA) inhibition profiling. The ethanol extract was fractionated by polarity. The ethyl acetate (EA) fraction, which showed the most potent inhibition of bacterial NA (NAC) (IC50 = 1.16 ± 0.18 µg/mL), was subjected to high-resolution bioactivity profiling. A total of 172 sub-fractions were assessed for NAC inhibitory activity. Active compounds were isolated based on their activity and identified using UPLC-MS/MS and NMR techniques. The inhibitory effects on both bacterial and viral neuraminidases (H3N2, H5N1 wild-type/mutant), as well as anti-H1N1 activity, were evaluated. Molecular docking and dynamics studies were conducted to elucidate the binding mechanisms. Twelve compounds outperformed Oseltamivir against NAC, with galloylated flavonoid glycosides exhibiting high potency (IC50 < 10 µM). Notably, all compounds demonstrated efficacy against both wild-type and Oseltamivir-resistant (H274Y mutant) viral neuraminidases. Galloyl-substituted flavonoid glycosides were identified as the principal active constituents. Although the isolated compounds exhibited only moderate inhibition of the H1N1 strain, the EA fraction showed stronger antiviral potency, with an EC50 of 248.3 ± 35.55 µg/mL. Molecular dynamics simulations confirmed stable binding of the active scaffolds to NA. Therefore, this study provides a chemical and molecular mechanistic basis for considering S. dulcificum leaves as a potential edible resource worthy of further investigation for its anti-influenza virus neuraminidase activity.