Structural investigations on betacyanin pigments by LC NMR and 2D NMR spectroscopy Florian C. Stintzing a , Ju¨rgen Conrad b, *, Iris Klaiber b , Uwe Beifuss b , Reinhold Carle a a Institute of Food Technology, Section Plant Foodstuff Technology, Hohenheim University, Garbenstrasse 25, D-70599 Stuttgart, Germany b Institute of Chemistry, Section Bioorganic Chemistry, Hohenheim University, Garbenstrasse 30, D-70599 Stuttgart, Germany Received 22 July 2003; received in revised form 10 October 2003 Abstract Four betacyanin pigments were analysed by LC NMR and subjected to extensive NMR characterisation after isolation. Pre- viously, low pH values were applied for NMR investigations of betalains resulting in rapid degradation of the purified substances thus preventing extensive NMR studies. Consequently, up to now only one single 13 C NMR spectrum of a betalain pigment, namely that of neobetanin (=14,15-dehydrobetanin), was available. Because of its sufficient stability under highly acidic conditions otherwise detrimental for betacyanins, this pigment remained an exemption. Since betalains are most stable in the pH range of 5–7, a new solvent system has been developed allowing improved data acquisition through improved pigment stability at near neutral pH. Thus, not only 1 H, but for the first time also partial 13 C data of betanin, isobetanin, phyllocactin and hylocerenin isolated from red-purple pitaya [Hylocereus polyrhizus (Weber) Britton & Rose, Cactaceae] could be indirectly obtained by gHSQC– and gHMQC–NMR experiments. # 2003 Elsevier Ltd. All rights reserved. Keywords: Betalains; Betacyanin; Betanin; Isobetanin; Phyllocactin; Hylocerenin; Pitaya; Hylocereus polyrhizus; NMR spectroscopy; LC NMR spectroscopy 1. Introduction Betalains are the nitrogenous vacuolar pigments of 13 families within the plant kingdom also accumulating in some members of the Basidiomycetes (Gill and Steglich, 1987; Gill, 1994; Clement and Mabry, 1996). They comprise two subgroups, the red-violet betacyanins and the yellow-orange betaxanthins (Steglich and Strack, 1990; Strack et al., 2003). Betalains are important chemotaxonomical markers and have never been found jointly with anthocyanins in the same plant (Stafford, 1994; Clement and Mabry, 1996). Besides their colour- ing function, betacyanins and betaxanthins exert diverse biological activities both in plants and in humans (Stintzing and Carle, 2004). Because of their high tinctorial strength and their colour hue remaining unchanged over a broad pH range from 3 to 7, beta- lains have attracted technological interest as natural food colourants (Von Elbe, 1975; Cai et al., 1998, 2001a; Cai and Corke, 1999; Stintzing et al., 2000, 2003). The structures of betalains have mainly been studied to shed light on biochemical pathways (Hempel and Bo¨hm, 1997; Schliemann et al., 1999; Kobayashi et al., 2000, 2001), but also to identify the colouring principles from plant tissues of various fruits and vegetables (Wyler and Dreiding, 1961; Gla¨ ssgen et al., 1993; Schliemann et al., 1996, 2001; Cai et al., 2001a,b; Stintzing et al., 2002a,b). The betacyanins from the red-violet cactus fruit [Hylocereus polyrhizus (Weber) Britton & Rose] also known as pitaya have been studied only recently by LC– MS and 1 H NMR investigations (Wybraniec et al., 2001; Stintzing et al., 2002b). However, no 13 C NMR data of the newly identified betacyanin, named hylocer- enin, were provided (Wybraniec et al., 2001). Yet with- out exception highly acidic conditions were applied for NMR spectra acquisition of betacyanins (Strack et al., 1993; Strack and Wray, 1994) and betaxanthins (Strack et al., 1987a). Since the betalains are labile under these conditions (Schliemann et al., 1996; Kobayashi et al., 0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2003.10.029 Phytochemistry 65 (2004) 415–422 www.elsevier.com/locate/phytochem * Corresponding author. Tel.: +49-711-459-2944; fax +49-711- 459-2951. E-mail address: chemconn@uni-hohenheim.de (J. Conrad).