Oxidation of oleuropein studied by EPR and spectrophotometry

Abstract

AbstractThe autoxidation at alkaline pH and enzymatic oxidation by mushroom tyrosinase of oleuropein, the dominant biophenol present in the fruits and leaves of Olea europea, was followed by both electron paramagnetic resonance (EPR) and absorption spectroscopy. For comparison, the same oxidation processes were applied to 4‐methylcatechol, a simple polyphenol present in olive mill wastewaters. EPR spectra of stable o‐semiquinone radicals produced during autoxidation at pH 12 and short‐lived o‐semiquinone free radicals produced during autoxidation at pH 9.0 or tyrosinase action and stabilized by chelation with a diamagnetic metal ion (Mg2+) were recorded for both polyphenols, and the corresponding hyperfine splitting constants were determined. The UV‐Vis spectral characteristics of the oxidation of polyphenols were highly dependent on the type of polyphenol, oxidant type and the pH of the reaction. The kinetic behavior of tyrosinase in the presence of oleuropein and 4‐methylcatechol was followed by recording spectral changes at 400 nm (absorption maximum) over time. The tysosinase activity with oleuropein showed a pronounced pH optimum at pH 6.5 and a minor one around pH 8. From the data analysis of the initial rate at pH 6.5, the kinetic parameters Km = 0.34 ± 0.03 mM and Vmax = 0.029 ± 0.002 ΔA400 min–1 were determined for oleuropein.