Oxidation of the flavonol fisetin by polyphenol oxidase

Abstract

The present study demonstrates the antiradical efficiency of fisetin, a flavonol widely distributed in fruits and vegetables, by its ability to react with two different free radicals, ABTS +⋅ and DPPH ⋅. The polyphenolic nature of fisetin led us to consider whether it might be oxidised by polyphenol oxidase (PPO), and the results reported show that it can be oxidised by PPO extracted and partially purified from broad bean seeds. The reaction was followed by recording spectral changes with time, with maximal spectral changes being observed at 282 nm (increase in absorbance) and at 362 nm (decrease). The presence of two isosbectic points (at 265 and 304 nm) suggested that only one absorbent product was formed. These spectral changes were not observed in the absence of PPO. The oxidation rate varied with the pH, reaching its highest value at pH 5.5. The fisetin oxidation rate increased in the presence of sodium dodecyl sulfate, an activator of polyphenol oxidase. Maximal activity was obtained at 0.87 mM sodium dodecyl sulfate. The following kinetic parameters were determined: V max=49 μM/min, K m=0.6 mM, V max/ K m=8.2×10 −2 min −1. Flavonol oxidation was inhibited by selective PPO inhibitors such as cinnamic acid (a classical competitive inhibitor, K i=1.4 mM) and 4-hexylresorcinol, which behaved as a slow-binding inhibitor. The results reported show that fisetin oxidation was strictly dependent on the presence of polyphenol oxidase.