Analytical Methods Identification of natural epimeric flavanone glycosides by NMR spectroscopy Federica Maltese a , Cornelis Erkelens b , Frank van der Kooy a , Young Hae Choi a, * , Robert Verpoorte a a Division of Pharmacognosy, Section Metabolomics, Institute of Biology, Leiden University, The Netherlands b Division of NMR, Leiden Institute of Chemistry, Leiden University, The Netherlands article info Article history: Received 17 September 2008 Received in revised form 17 January 2009 Accepted 1 March 2009 Keywords: Flavanone Citrus fruits Stereoisomers NMR abstract Recently advanced analytical technology has provided evidence of the existence of stereoisomers of many natural products. Particularly, flavanones which might have two different configurations at C-2 exist in many food additives, e.g., citrus fruits. In this study, the possible stereoisomers of flavanone glycosides were identified by NMR spectroscopy. Based on NMR spectra of common flavanone glycosides such as naringin, hesperidin, and neohesperidin, the two existing diastereomeric forms of the molecules could clearly be distinguished. The 1 H NMR resonances of two diastereomers of each flavanone glycosides investigated in this study were fully assigned with the assistance of diverse 2D NMR spectroscopy methods. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Flavonoids represent one of the largest plant secondary metab- olite groups with more than 6500 reported compounds up to the year of 2000. Flavonoids are largely present in our daily life, ingested via fruits, vegetables, legumes, and medicinal herbs. The importance of this huge class of plant phenolic constituents lies in their wide range of biological and physiological roles, mostly attributed to health benefits such as antioxidant, anti-inflamma- tory, oestrogenic, antiviral, and chemopreventive properties (Harborne & Williams, 2000). Flavonoids are grouped in several subclasses, such as anthocya- nins, flavans, isoflavonoids, flavones, flavanones, flavonols, and catechines, all of them chemically characterized by a 2-phenyl- benzopyran-4-one structure. In particular the class of flavanones is so widely occurring in citrus fruits that several authors have cho- sen natural flavanones as chemotaxonomic markers and for the identification of adulterated processed juices (Albach & Redman, 1969; Kefford, 1959; Ooghe, Ooghe, Detavernier, & Huyghebaert, 1994; Wistuba, Trapp, Gel-Moreto, Galensa, & Schurig, 2006). Among all the other flavonoids, the subclass of flavanones presents the unique characteristic of possessing a chiral center at the C-2 position, which potentially leads to the existence of two stereoisomeric forms (Fig. 1). Moreover, like other flavonoids, flav- anones generally possess sugars attached to various –OH groups. The resulting changes of configuration at the stereogenic centers of the molecule determine that flavanone glycosides may exist as mixtures of diastereoisomers. The determination of the isomeric composition of natural prod- ucts in food products has received great attention for a long time. Positive bioactivities might be lost or transformed into undesirable toxicity or inactivity by the formation of stereoforms of natural compounds (Marder et al., 2003). It is believed that the involve- ment of a manifold of enzymes in the biosynthesis of phytochemi- cals results in the highly stereoselective production of the final natural products. However, this monolithic pressumption is now being challenged. In fact, recent advances in modern analytical technology used for the reassessment of formerly described natu- ral products structures have allowed the discovery of natural ste- reoforms. In many cases, compounds which were believed to be present as one specific stereoform in nature were reviewed and found to occur as stereoisomers. NMR spectroscopy has been rec- ognized so far as the most important tool for the structural eluci- dation of flavonoids (Fossen & Andersen, 2006) and it is also increasingly used as an analytical tool for the evaluation of quality traits in foods (Le Gall, Puaud, & Colquhoun, 2001; Vogels et al., 1996). Together with its undisputed potential in structural elucida- tion, NMR has other advantages such as a quick sample preparation and short analysis time, and its total sample recovery. Additionally, it also allows the analysis of stereoisomeric mixtures. Gaffield, Lundin, Gentili, and Horowitz (1975) used 1 H NMR as supporting material to circular dichroism measurements for an estimation of the isomeric composition of naringin in grapefruit at different rip- ening stages. The study allowed the authors to conclude that the (2S)-isoform of naringin in grapefruit, the main form present in premature fruits, undergo to racemization in maturing grapefruit. The isomeric composition of commercial enzymatically modified naringin used as food additive was also analysed by Akiyama, Yam- ada, Yamada, and Maitani (2000) by means of 1 H NMR and 13 C 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.03.023 * Corresponding author. Tel.: +31 71 527 4471; fax: +31 71 527 4511. E-mail address: y.choi@chem.leidenuniv.nl (Y.H. Choi). Food Chemistry 116 (2009) 575–579 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem