An EPR study of free radical generation during maceration of uncooked vegetables Bernard A Goodman,* Sheila M Glidewell, Catriona M Arbuckle, Sylvie Bernardin, Timothe ´e R Cook and John R Hillman Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Abstract: Electron paramagnetic resonance (EPR) spectroscopy has been used to study free radicals formed in a range of plant tissues as a result of physical damage, with the objective of gaining some insight into the free radical chemistry that is initiated when uncooked vegetable products (eg salads) are eaten. Chemical spin traps were used to aid the detection of unstable free radicals; more stable radicals were detected directly. Commonly observed ‘stable’ species were the monodehydroascorbate radical, which has a characteristic doublet spectrum, and a single-peak resonance, which is presumed to come from free radical centres stabilised in macromolecules. In mushrooms (Agaricus spp), spin- trapping experiments using either a-(4-pyridyl-1-oxide)-N-t-butyl-nitrone (4-POBN) or phenyl-t- butyl-nitrone (PBN) showed the formation of large quantities of adducts of the radical from 4- hydroxymethylbenzene diazonium salts. Pleurotus species, in contrast, gave signals consistent with the formation of unidentified C-centred radicals. With other foodstuffs, reaction with 4-POBN was complex and signals from 4  POBN . and CO  2 adducts were observed along with the t-butylhydro- nitroxide radical (an adduct breakdown product). Investigation of carrot hypocotyl rootstock in the presence of 5-(diethoxyphosphoryl),5-methyl-1-pyrroline-N-oxide (DEPMPO) revealed adducts of . OH and unidentified C-centred radicals. Free radical interactions between food components were demonstrated by the suppression of the signal from the 4-POBN adducts of lettuce by onion, garlic, satsuma or vinaigrette, but not by olive oil. In addition, an appreciable decrease in spectral intensity of the 4-POBN adduct from lettuce was observed in the presence of saliva, which suggests that saliva contains free radical scavengers which are able to compete successfully with the spin trap. # 2002 Society of Chemical Industry Keywords: free radicals; EPR spectroscopy; spin trapping; vegetables; saliva INTRODUCTION The consumption of diets containing substantial quantities of fruit and vegetables is associated with reduced risks for the development of several types of cancer 1,2 and heart disease. 3,4 Oxygen-derived free radicals have been implicated in many disease pro- cesses, 5 and Bolton-Smith et al 6 have shown that there is an inverse relationship between the intake of antioxidant vitamins and the risk of coronary heart disease, particularly in men. Consequently, consider- able scientific activity is being aimed at evaluating the protective modes of action of vitamins and other antioxidant molecules on mammalian cells, 7 and dietary supplementation is now common in developed countries. Plants represent a major source of vitamins and other antioxidants such as polyphenols in unsupple- mented diets, and there is now considerable interest in the genetic manipulation of crops in order to design foods and diets for optimal health effects. 8 In addition to the production of enhanced levels of selected vitamins, maximisation of the total antioxidant capa- city of foods is becoming recognised as an important quality criterion. 9 In addition to the production of antioxidant mol- ecules as aids to survival in an aerobic environment under solar radiation, plants make use of free radicals in normal metabolic processes such as photosynth- esis 10 and in their defences against (potentially) pathogenic organisms and predators. 11 In the latter case, free radical production is initiated in response to cellular damage and, consequently, similar reactions should occur when the product is eaten. Little attention has, however, been given to the generation of free radicals during food consumption, yet these free radical reactions might be expected to occur to a substantial extent during the digestive process and may also be relevant to diseases of the digestive tract. The present paper reports observations using EPR spectroscopy to investigate free radical generation on maceration of a range of vegetable products that are commonly consumed in an uncooked state. The mini- (Received 4 October 2001; revised version received 25 March 2002; accepted 16 April 2002) * Correspondence to: Bernard A Goodman, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK E-mail: bgoodm@scri.sari.ac.uk Contract/grant sponsor: Scottish Executive Environment and Rural Affairs Department # 2002 Society of Chemical Industry. J Sci Food Agric 0022–5142/2002/$30.00 1208 Journal of the Science of Food and Agriculture J Sci Food Agric 82:1208–1215 (online: 2002) DOI: 10.1002/jsfa.1180