ESR studies of vitamin C regeneration, order of reactivity of natural source phytochemical preparations.

The effect of various flavonoids upon the ascorbate radical lifetime was investigated by ESR spectroscopy. The radical was generated via the reaction between ascorbic acid and ascorbate oxidase, the ascorbate radical being detected. The inclusion of the flavonoids in the ascorbic acid-ascorbate oxidase reaction mixture affected both the initial intensity of the ascorbate radical and its lifetime. Of the natural sources tested, Pycnogenol prolonged the ascorbate radical lifetime to the greatest extent, from a control value of 20 min to a maximum of 80 min with 200 micrograms/ml Pycnogenol. The flavonoids could either be regenerating ascorbic acid from ascorbate, or interacting with ascorbate oxidase, thus preventing ascorbic acid binding. When p-cresol, a known ascorbate oxidase inhibitor was added to the ascorbic acid-ascorbate oxidase reaction mixture, the ascorbate radical signal intensity was dramatically reduced and did not display the time-dependent decay observed with the flavonoids. This indicates that a direct interaction between the flavonoids and ascorbate radical occurs. Some of the flavonoids tested; myricetin, polyphenon and theaflavin appeared to compete with ascorbic acid for ascorbate oxidase, as they displayed saturation behaviour. By modifying the experimental conditions the myricetin radical was detected, thus confirming the direct interaction between myricetin and ascorbate oxidase.