Ortho nitrophenyl β d galactopyranoside

Glucosinolate contents were determined according to the previous report with minor modifications [14 (link)]. In this study, samples were boiled in 5 mL water for 10 min, and then the supernatant was transferred to a new tube after centrifugation (5 min, 7000× g). The residues were boiled for another 10 min with water (5 mL). The combined supernatants were applied to a DEAE-Sephadex A-25 column, and the glucosinolates were purified and converted to desulphoglucosinolates, as described [14 (link)]. Samples were subjected to the high-performance liquid chromatography (HPLC) analysis by using an HPLC instrument (Shimadzu, Kyoto, Japan) with an SPD-M20A diode array detector. A hypersil C18 column (5 μm particle size, 4.6 mm × 250 mm; Elite Analytical Instruments Co. Ltd., Dalian, China) was used with a mobile phase of acetonitrile and water at a flow rate of 1 mL/min. The procedure employed isocratic elution with 1.5% acetonitrile for the first 5 min; a linear gradient to 20% acetonitrile over the next 15 min; followed by isocratic elution with 20% acetonitrile for the final 13 min. Absorbance was measured at 226 nm. The Ortho-nitrophenyl-β-d-galactopyranoside (Sigma, St. Louis, MO, USA) was used as an internal standard for HPLC analysis. Data were expressed as μmol/g dry weight (DW).

Glucosinolates were extracted and analyzed as previously described (1 (link)). Freeze-dried samples (100 mg) were boiled in 5 mL water for 10 min. The supernatant was collected after centrifugation, and the residues were washed once with water, centrifuged, and then combined with the previous extract. The aqueous extract was applied to a DEAE-Sephadex A-25 column (Sigma Chemical Co., Saint Louis, USA). The glucosinolates were converted into their desulpho analogs by overnight treatment with 100 μL of 0.1% aryl sulphatase (Sigma Chemical Co., Saint Louis, USA), and the desulphoglucosinolates were eluted with 1 mL water. HPLC analysis of desulphoglucosinolates was carried out using an Agilent 1260 HPLC instrument equipped with a VWD detector (Agilent Technologies, Inc., Palo Alto, USA). Samples were separated at 30°C on a Waters Spherisorb C18 column (250 × 4.6 mm i.d.; 5 μm particle size) using acetonitrile and water at a flow rate of 1.0 mL min⁻¹. Absorbance was detected at 226 nm. Glucosinolates were quantified by using ortho-Nitrophenyl β-D-galactopyranoside (Sigma Chemical Co., Saint Louis, USA) as the internal standard and considering the response factor of each glucosinolate. Result of glucosinolate content was expressed as μmol g⁻¹ of dry weight.