Competitive neutrophil elastase inhibitory isoflavones from the roots of <i>Flemingia philippinensis</i>

다국어 초록 (Multilingual Abstract)

<P><B>Abstract</B></P> <P> <I>Flemingia philippinensis</I> has been used throughout history to cure rheumatism associated with neutrophil elastase (NE). In this study, we isolated sixteen NE inhibitory flavonoids (<B>1</B>–<B>16</B>), including the most potent and abundant prenyl isoflavones (<B>1</B>–<B>9</B>), from the <I>F. philippinensis</I> plant. These prenyl isoflavones (<B>2</B>, <B>3</B>, <B>5</B>, <B>7</B>, and <B>9</B>) competitively inhibited NE, with IC<SUB>50</SUB> values of 1.3–12.0 μM. In addition, they were reversible, simple, slow-binding inhibitors according to their respective parameters. Representative compound <B>3</B> had an IC<SUB>50</SUB> = 1.3 μM, <I>k</I> <SUB>3</SUB> = 0.04172 μM<SUP>−1</SUP> min<SUP>−1</SUP>, <I>k</I> <SUB>4</SUB> = 0.0064 min<SUP>−1</SUP>, and <I>K</I> <SUB>i</SUB> <SUP>app</SUP> = 0.1534 μM. The <I>K</I> <SUB>ik</SUB>/<I>K</I> <SUB>iv</SUB> ratios (18.5 ∼ 24.6) for compound <B>3</B> 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 (<B>10</B>–<B>16</B>) 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 (IC<SUB>50</SUB>) closely followed the binding affinity (<I>K</I> <SUB>SV</SUB>).</P> <P><B>Highlights</B></P> <P> <UL> <LI> Phenolic isolates from <I>Flemingia philippinensis</I>. </LI> <LI> <I>In vitro</I> neutrophil elastase inhibitory activity. </LI> <LI> Characterization of kinetics based on structure. </LI> <LI> Binding affinity between inhibitor and enzyme. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>