Analytical Methods Development of a reliable extraction and quantification method for glucosinolates in Moringa oleifera Nadja Förster a , Christian Ulrichs a , Monika Schreiner b , Carsten T. Müller c , Inga Mewis b,⇑ a Division Urban Plant Ecophysiology, Humboldt-Universität zu Berlin, Lentzeallee 55-57, 14195 Berlin, Germany b Department Quality, Leibniz-Institute of Vegetable and Ornamental Crops, Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germany c School of Biosciences, Cardiff University, The Sir Martin Evans Building, Museum Avenue, Cardiff CF10 3AX, UK article info Article history: Received 23 July 2013 Received in revised form 18 February 2014 Accepted 8 June 2014 Available online 14 June 2014 Keywords: Glucosinolate Intact extraction method Desulfo extraction method Moringa oleifera abstract Glucosinolates are the characteristic secondary metabolites of plants in the order Brassicales. To date the common DIN extraction ‘desulfo glucosinolates’ method remains the common procedure for determina- tion and quantification of glucosinolates. However, the desulfation step in the extraction of glucosino- lates from Moringa oleifera leaves resulted in complete conversion and degradation of the naturally occurring glucosinolates in this plant. Therefore, a method for extraction of intact Moringa glucosinolates was developed and no conversion and degradation of the different rhamnopyranosyloxy-benzyl glucosin- olates was found. Buffered eluents (0.1 M ammonium acetate) were necessary to stabilize 4-a-rhamno- pyranosyloxy-benzyl glucosinolate (Rhamno-Benzyl-GS) and acetyl-4-a-rhamnopyranosyloxy-benzyl glucosinolate isomers (Ac-Isomers-GS) during HPLC analysis. Due to the instability of intact Moringa gluc- osinolates at room temperature and during the purification process of single glucosinolates, influences of different storage (room temperature, frozen, thawing and refreezing) and buffer conditions on glucosin- olate conversion were analysed. Conversion and degradations processes were especially determined for the Ac-Isomers-GS III. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introductory statement Glucosinolates are one of the most investigated groups of secondary metabolites in plants. They are of crucial importance in insect–plant interactions and can act as feeding deterrent (gen- eralists) or as attractant (specialists) (Mewis, Khan, Glawischnig, Schreiner, & Ulrichs, 2012). Additionally, glucosinolates have the ability to influence human health as phytopharmaceutical, nutra- ceutical, or as food product (Melchini et al., 2013; Prakash & Gupta, 2012). Glucosinolates are a group of secondary plant metab- olites found almost exclusively in plants of the order Brassicales. Structurally glucosinolates are b-S-glucosides of thio-oxime-O-sul- phates and synthetized from amino acids. They are commonly divided into aliphatic, indolic, and aromatic glucosinolates based on their side chains, which stem from the precursor amino acids methionine, tryptophan and phenylalanine respectively. Profile and abundance of glucosinolates vary between different parts of a plant. For example, roots of over 29 analysed plant species (mainly members of the Brassicaceae) contained significantly higher aromatic glucosinolate levels than shoots (van Dam, Tytgat, & Kirkegaard, 2009). Aromatic glucosinolates such as p-hydroxybenzyl glucosinolate (sinalbin), 2-phenylethyl glucosin- olate (gluconasturtiin), benzyl glucosinolate (glucotropaeolin), or 2(R)-hydroxy-2-phenylethyl glucosinolate (glucobarbarin) dominated in seeds and roots of white mustard (Sinapsis alba), watercress (Barbarea verna), Indian mustard (Tropaeolum majus), or winter cress (Barbarea vulgaris)(Bennett, Mellon, & Kroon, 2004; van Leur, Raaijmakers, & van Dam, 2006). The family Mor- ingaceae (order Brassicales) includes only the genus Moringa. In contrast to other Brassicales, all species of Moringa, and particu- larly Moringa oleifera, contain very high amounts of aromatic gluc- osinolates in all parts of the plant including leaves and roots, which overall showed lower levels of glucosinolates than seeds (seeds around 200 mg/g DW, young leaves around 116 mg/g DW, older leaves around 63 mg/g DW, roots around 43 mg/g DW, Bennett et al., 2003). Structurally, all multiglycosylated aromatic glucosinolates con- tain a hydroxy-benzyl moiety with a sugar molecule – rhamnose or arabinose – linked glycosidically to the aromatic ring. This gener- ally appears to be unique to species of the genera Moringa, Hesperis, and Reseda (Bennett et al., 2003; Fahey, Zalcmann, & Talalay, 2001). 4-a-rhamnopyranosyloxy-benzyl glucosinolate (Rhamno-Benzyl- GS) has been identified as the dominant leaf glucosinolate of http://dx.doi.org/10.1016/j.foodchem.2014.06.043 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +49 033701 78 244; fax: +49 033701 78 5250. E-mail address: inga@entomology.de (I. Mewis). Food Chemistry 166 (2015) 456–464 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem