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GENERAL DISCUSSION
Carrots, herbs and mushrooms were investigated by grinding a small amount of the material in the presence of a spin trap to detect free radical formation generated by cell disruption. With mushroom, a trapped carbon-centred radical was identified as 4-(hydroxymethyl)phenyl radical, whereas with carrots and herbs the spin adducts were assigned to the .OH radical and carbon-centred radicals in general but with no specific identification. The 4-(hydroxymethyl)benzene diazonium salt which is the substrate for the 4-(hydroxymethyl)phenyl radical is found in appreciable amounts in mushrooms (Levenberg, 1962; Ross et al., 1982) .
Herb samples were examined in a screening experiment using the spin trap 4-POBN. A few spectra of herbs were dominated by the ascorbate radical, some showed a spin adduct similar to a carbon-centred radical, but the majority of the investigated herb extracts were able to oxidise the spin trap. This oxidation product of the spin trap 4-POBN was also seen with carrot samples in addition to t-butylhydronitroxide, a breakdown product of the spin traps PBN and 4-POBN. It's possible that this pro-oxidant activity arises from specific plant components which are oxidised by cell disruption under atmospheric condition. The resulting product(s) may be powerful oxidants which are able to oxidise and degrade the spin trap. Carrot samples taken from different positions of the root (top, centre, tip) varied in their pro-oxidative activity showing the highest activity in the tip of the root which may be correlated with a higher generation of .OH radicals in this growing zone (Liszkay et al., 2004; Schopfer et al., 2002; Schopfer, 2001). Variations in the pro-oxidative behaviour were also observed with different herb leaves of sage which were harvested on different times on a day distinguishing between sunny and shady leaves. However, inconsistencies in the results with the sage leaves made it impossible to relate them to time or sunlight intensity.
Different leaves showed randomly either the presence of a carbon-centred radical or oxidised the spin trap 4-POBN.
This pro-oxidative ability of tissue extracts from carrots and herbs was the basis for further investigations of oxidation reactions with specific phenolic compounds
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(kaempferol, luteolin, rosmarinic acid (RA) and camosic acid (CA» which occur in a significant amount in herbs (kaempferol in lemon balm; luteolin in parsley, 'thyme, peppermint, basil; RA and CA in rosemary and sage). These reactions were initiated with 5 different systems - autoxidation at -pH 13, HRPIH202, X/XO, Fenton reaction system and K02 at pH 7.
The .OH radical which is often the initial radical formed in living systems e.g. by the Fenton reaction, is not stable and reacts with virtually every molecule it contacts. Itis often the initiator of lipid peroxidaton by abstracting a hydrogen atom from lipid molecules such as fatty acids. (Halliwell and Gutteridge, 1984) Since it plays an important role in biochemical reactions it was chosen as oxidising agent in a Fenton-like reaction system.
Superoxide anion radicals which are major products of the photosynthetic pathway (Smimoff, 1993), and therefore are present in any green tissue, were taken as the second oxidation medium. They were generated using the enzyme system xanthine/xanthine oxidase and potassium superoxide, both at pH 7.
Phenolic compounds were also oxidised with a solution containing the enzyme HRP and H202, which functions by generating compounds with iron in a high (+IV) oxidation state. Compound I is reduced to compound II by oxidising a phenolic compound. This reaction is repeated with compound II and oxidation of another phenolic molecule leads to regeneration of the original enzyme structure. (Chen and Schopfer, 1999)
Plants protect themselves against free radical damage by the production of antioxidant molecules. In herbs this role is often ascribed to phenolic compounds such as flavonoids. (Pietta et al., 2003) The antioxidant activity of such compounds has been investigated extensively (Sichel et al., 1991; Pedrielli et al., 2001; McPhail et al., 2003;
Bors and Saran, 1987), although the in vivo function of flavonoids is still not clare.
There is evidence that flavonoids not only function as antioxidants, but they may
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supplementation of flavonoids (Skibola and Smith, 2(00), and the influence and consequence of the radical formation of flavonoids is not fully understood, even though their chemistry has been studied over many years.
This work presents some new EPR spectra of oxidised phenolic compounds. The stability of the phenolic radicals ranged from -6 min. (semiquinone radical generated by the oxidation of kaempferol) to >3 hours (hydroxylated structure of RA).
Autoxidation which plays an important role in food storage processes, was used to gain preliminary information about the radical formation of such antioxidants. Some spectra from the autoxidation experiments at pH 13 were also observed with oxidation conditions at pH 7, which are physiologically more relevant.
Although the flavonoids luteolin and kaempferol differ only in the position of one hydroxyl group, they behaved completely differently under the applied oxidation conditions. Whereas kaempferol reacted with the formation of degradation products and dimers, the structure of luteolin was sustained after oxidation. Various measurements of the antioxidant activity did not produce a clear picture as to which of the two compounds has the higher free radical scavenging activity, e.g. peroxyl radicals and azide radicals were better scavenged by kaempferol, but galvinoxyl radicals were scavenged from both flavonoids with equal intensity (Madsen et al., 2000; McPhail et al., 2003; Bors and Saran, 1987). The sustained free radical scavenging activity of luteolin is probably due to its ability to redox cycle, whereas the activity of kaempferol may be related to the chemistry of its metabolites.
RA and CA are main components in rosemary and sage and are thought to be also responsible for some of the positive health effects of these herbs (Munné-Bosch and Alegre, 2001; del Bano et al., 2003). In both structures catechol groups are the sites of oxidation. It seems as if the original structure of RA remains intact after oxidation as is the case with luteolin. EPR spectra of luteolin and RA, recorded after some time in alkaline solution, are most likely based on hydroxylated structures. CA is not regenerated once it is oxidised. Monné-Bosch et al. (2001) made concentration measurements in cell compartments and showed that the location of CA and its oxidation product carnosol is only at their site of generation, in chloroplasts. After
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oxidation the concentration of other products such as rosmanol and isorosmanol increased in the cells. The present EPR results indicate free radical formation from both CA and camosol.
All of the chosen phenolic compounds could be oxidised and free radicals were detected under the various oxidation conditions. The EPR signals were sufficiently stable to produce good quality spectra, most of which could be interpreted, and some structures could be formulated. The concentrations of the phenolic solutions in these measurements were between 0.5 and I mM, which may be already in the range for toxicity effects according to some in vitro experiments (Spencer et al., 2003), but the results may still serve as basis for further investigations under physiological relevant conditions .
Itcould be clearly shown that the mechanism of the reaction of antioxidants and free radicals varies with the antioxidant molecule, the type of oxidising reagent and pH.
These results indicate the importance of such EPR measurements in addition to biochemical methods for understanding antioxidant behaviour.