Studies on Nonenzymatic Oxidation Mechanisms in Neobetanin, Betanin, and Decarboxylated Betanins Slawomir Wybraniec,* ,† Karolina Starzak, † Anna Skopiń ska, † Boris Nemzer, ‡ Zbigniew Pietrzkowski, § and Tadeusz Michalowski † † Department of Analytical Chemistry, Institute C-1, Faculty of Chemical Engineering and Technology, Cracow University of Technology, ul. Warszawska 24, Cracow 31-155, Poland ‡ Chemistry Research, FutureCeuticals, Inc., 2692 North State Route 1-17, Momence, Illinois 60954, United States § Applied BioClinical Inc., 16259 Laguna Canyon Road, Irvine, California 92618, United States ABSTRACT: A comprehensive nonenzymatic oxidation mechanism in betanin plant pigment as well as its derivatives, 2- decarboxybetanin, 17-decarboxybetanin, 2,17-bidecarboxybetanin, and neobetanin, in the presence of ABTS cation radicals was investigated by LC-DAD-ESI-MS/MS. The main compounds formed during the first step of betanin and 2-decarboxybetanin oxidation are 2-decarboxy-2,3-dehydrobetanin and 2-decarboxyneobetanin, respectively. In contrast to betanin, the reaction mechanism for 2-decarboxybetanin includes more oxidation pathways. Parallel transformation of 2-decarboxybetanin quinone methide produces neoderivatives according to an alternative reaction that omits the presumably more stabile intermediate 2- decarboxy-2,3-dehydrobetanin. The main oxidation product after the first reaction step for both 17-decarboxybetanin and 2,17- bidecarboxybetanin is 2,17-decarboxy-2,3-dehydrobetanin. This product is formed through irreversible decarboxylation of the 17- decarboxybetanin quinone methide or by oxidation of 2,17-bidecarboxybetanin. Oxidation of neobetanin results primarily in a formation of 2-decarboxy-2,3-dehydroneobetanin by a decarboxylative transformation of the formed neobetanin quinone methide. The elucidated reaction scheme will be useful in interpretation of redox activities of betalains in biological tissues and food preparations. KEYWORDS: betanin, neobetanin, betacyanins, dopachrome, aminochrome, quinone methide, antioxidation, 5,6-dihydroxyindole ■ INTRODUCTION Betalains are a group of water-soluble plant pigments that are used in industry as food colorants 1,2 and possess chemo- preventive and strong antioxidant properties. 3-6 The betalain subgroup includes betacyanins (Figure 1), which are primarily immonium conjugates of betalamic acid with glycosylated cyclo- DOPA . 1,2,7 Betanin 1, the principal pigment of red beet root (Beta vulgaris L.), was the first and most frequently studied betalain for its antioxidant activity. 3,5,6,8-13 Among others, the isolated betacyanins were tested with 1,1-diphenyl-2-picrylhy- drazyl (DPPH) 11 and 2,2′-azinobis(3-ethylbenzthiazoline-6- sulfonic acid) (ABTS) 12,13 radicals. The antioxidant activity of purified betalains against ABTS has been tested under the influence of pH and other physicochemical conditions to explore structure activity relationships in betalains 12,13 and in a particularly important betalain structure constituent, betalamic acid. 14 Although recent enzymatic studies 15 have shed some light on the oxidation pathways of betacyanins, the nonenzymatic structural data are still lacking. Recent studies on the structural implications of semisynthetic natural or artificial betalains confirmed the fact that the high antioxidant activity of these molecules is affected by the presence of one or two phenolic groups, but raised a possibility that other unknown structural factors should also be taken into account. 12-14 With the exception of the enzymatic 15 and electrochemical 16 research on betalains, the mechanism by which ABTS radicals oxidize betalains is also unknown. Understanding the structural features of these pigments that are responsible for their antioxidant effect has great bearing on future development in this area. ABTS radicals are very commonly used organic probes for evaluating the antioxidant activity of natural compounds. The kinetics of the reactions between the ABTS radical cations and several groups of compounds (e.g., polyphenols) are quite complex, and the lack of a relationship between the rate and stoichiometric factors has been reported. 17 In addition, some of the degradation products of ABTS radicals were identified, suggesting a lability of ABTS radicals at certain conditions. 18 In recent studies on the enzymatic oxidation of betanidin (deglucosylated betanin), 15 the presence of prominent oxidation products at pH 3, 2-decarboxy-2,3-dehydrobetanidin and 2,17- bidecarboxy-2,3-dehydrobetanidin, indicated their generation via two possible reaction paths with two different quinonoid intermediates: dopachrome and quinone methide derivatives. Both reaction pathways lead to the decarboxylative dehydrogen- ation of betanidin. Subsequent oxidation and rearrangement of the conjugated chromophoric system results in formation of 14,15-dehydrogenated derivatives. At higher pH (4-8), two main oxidation peaks of betanidin are observed: betanidin quinonoid (presumably betanidin o-quinone) and 2-decarboxy- 2,3-dehydrobetanidin. 15 In contrast, betanin (5-O-glucosylated Received: February 23, 2013 Revised: May 21, 2013 Accepted: May 22, 2013 Published: June 24, 2013 Article pubs.acs.org/JAFC © 2013 American Chemical Society 6465 dx.doi.org/10.1021/jf400818s | J. Agric. Food Chem. 2013, 61, 6465-6476