Determination of Glucosinolates in Plant Samples

The glucosinolates were extracted and determined according to a modified method of Heaney and Fenwick (1980) (link) and O’Callaghan et al. (2000) (link). Frozen plant material was freeze-dried in a LyoLAB 3000 freeze drier (Heto-Holten A/S, Allerød, Denmark) for 3 days. Freeze-dried plant samples were pulverized in a Retsch Mixer-Mill (Retsch type MM2; Haan, Germany). The glucosinolates were extracted in boiling 90% methanol (50 mg in 3 ml) for 2 min. The extract was centrifuged for 2 min at 2,500 g and the residue was re-extracted twice with 3 ml boiling 70% methanol. Total glucosinolate content was determined based on its reaction with sodium tetrachloropalladate II (Na₂PdCl₄; Gupta et al., 2012 (link); Ishida et al., 2012 (link)). The reaction mixture containing 60 μl extract and 1800 μl 2 mM Na₂PdCl₄ was incubated at 20°C for 30 min and the absorbance of the developed color measured colorimetrically at 450 nm (Thies, 1982 (link)). Sinigrin (Sigma–Aldrich, S1647) was used as an internal standard for all samples (13 μmol per extracts) and data were corrected for recovery rate (always higher than 80%).