Abstract
Flemingia philippinensis has been used throughout history to cure rheumatism associated with neutrophil elastase (NE). In this study, we isolated sixteen NE inhibitory flavonoids (1-16), including the most potent and abundant prenyl isoflavones (1-9), from the F. philippinensis plant. These prenyl isoflavones (2, 3, 5, 7, and 9) competitively inhibited NE, with IC50 values of 1.3-12.0 μM. In addition, they were reversible, simple, slow-binding inhibitors according to their respective parameters. Representative compound 3 had an IC50 = 1.3 μM, k3 = 0.04172 μM-1 min-1, k4 = 0.0064 min-1, and Kiapp = 0.1534 μM. The Kik/Kiv ratios (18.5 ∼ 24.6) for compound 3 were consistent with typical competitive inhibitors. The prenyl functionality of isoflavones significantly affected inhibitory potencies and mechanistic behavior by shifting the competitive mode to a noncompetitive one. The remaining flavonoids (10-16) were confirmed as mixed type I inhibitors that preferred to bind free enzyme rather than the enzyme-substrate complex. Fluorescence quenching analyses indicated that the inhibitory potency (IC50) closely followed the binding affinity (KSV).