Effect Of Natural Flavonoid Antioxidants On The Oxidation Of Benzidine And Other Xenobiotics Mediated By Soybean Lipoxygenase

[Objectives] This study is to investigate the effect of the natural flavonoid antioxidants (tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG) , quercetin and rutin) on the oxidation of four aromatic compounds (guaiacol, benzidine, p-phenylenediamine and o-dianisidine) mediated by lipoxygenase, and to search for some effective inhibitors of the oxidation. It would provide important evidence that anti-toxic and anti-tumor effect of natural antioxidant can be caused by a new mechanism of inhibiting the activation and oxidation of xenobiotics mediated by lipoxygenase. These would provide further proof that lipoxygenase is the alternative pathway of xenobiotic oxidation mediated by cytochrome P450.[Methods] On the in vitro suitable reaction conditions, soybean lipoxygenase(SLO), modifiers, substrates and so forth were added into reaction medium, and followed by recording an increase in the absorption with time at the specified wavelength and the different spectrum using UV/VIS7501 ultraviolet spectrophotometer, or detecting the free radical intermediate in the reaction medium using JES-FE1XG electron spin resonance spectrometer. With the oxidative rate of four aromatic compounds catalyzed by SLO under different conditions, the inhibitive rate and the 50% inhibitory concentration of these antioxidants for the oxidations were calculated, respectively.[Results] In the presence of hydrogen peroxide, SLO can catalyze the co-oxidation of guaiacol, benzidine, p-phenylenediamine and o-dianisidine. There are free radical intermediates to produce in the process of co-oxidation. Under optimal condition, Vmax value of the oxidation of guaiacol catalyzed by soybean lipoxygenase was 2.33nmol ?min-1 ?nmol-1SLO. The reaction exhibited Km value of 7.54mmol ?L-1 for guaiacol. The reductants reduced glutathion (GSH)and dithiothreitol (DTT) , free radical scavenger butylated hydroxyanisole (BHA), classical lipoxygenase inhibitors gossypol and natural flavonoid antioxidants tea polyphenol, quercetin and rutin can inhibit the oxidation of four aromatic compounds catalyzed by SLO, which appears to be concentration-dependent manner in special range. Under the optimal condition of the oxidation of guaiacol mediated by SLO, the 50% inhibition concentration of these natural antioxidants for the cooxidation (IC50) were significant lower than common modifiersof lipoxygenase activity ___GSH,DTT,BHA and gossypol.[Conclusions] Tea polyphenols and its EGCG monomer, quercetin and rutin can all obviously inhibit the oxidation of four aromatic compounds including guaiacol and benzidine catalyzed by SLO. The inhibition of them are more potent than GSH, DTT, BHA and gossypol. On the other hand, the inhibitive activity of tea polyphenols and EGCG are higher than quercetin and rutin. The inhibitive effect of natural antioxidant on oxidation and activation of xenobiotics mediated by lipoxygenase may be one of the mechanisms of its anti-toxic and anti-tumor effect.