Glucoiberin

The standards of sinapic and caffeoylquinic acids were purchased from Sigma-Aldrich (Steinheim, Germany). The standards of glucoraphanin, glucoerucin, glucoiberin, glucobrassicin, hydroxyl-glucobrassicin, gluconasturtiin, methoxy-glucobrassicin, neoglubobrassicin, 3,4-diindolylmethane, and iberin (GR, GE, GI, GB, HO-GB, PE, MeOH-GB, NeoGB, SFN, I3C, and DIM, respectively) were purchased from Phytoplan GmbH (Heidelberg, Germany). The standards of sulforaphane, iberin, and indole-3-carbinol (SFN, IB, and I3C, respectively) were obtained from Santa Cruz Biotechnology (Dallas, US), Biorbyt LTD (Cambridge, UK), and LKT Laboratories (St. Paul, MN, USA), correspondingly. Acetic and hydrochloric acids, as well as ammonium acetate, were from Panreac labs (Barcelona, Spain). Methanol for hydromethanolic extractions and acetonitrile and acetic acid grade solvents for LC-MS were supplied by J.T. Baker (Philipsburg, NJ, USA). All water employed in the extraction and the chromatographic analysis was purified by using a Milli-Q system (Millipore, Bedford, MA, USA).

The standards of GSLs, namely glucoiberin, glucoraphanin, hydroxy-glucobrassicin, glucoerucin, glucobrassicin, gluconasturtiin methoxy-glucobrassicin, and neo-glucobrassicin (GI, GR, HGB, GE, GB, PE, MGB, NGB, respectively), and the standards of ITC and indoles, namely D,L-sulforaphane L-cysteine, D,L-sulforaphane glutathione, erucin, D,L-sulforaphane-N-acetyl-L-cysteine, iberin, indole-3-carbinol, sulforaphane, and 3,4-diindolylmethane (SFN-CYS, SFN-GSH, E, SFN-NAC, IB, I3C, SFN, DIM, respectively) were obtained from Phytoplan GmbH (Heidelberg, Germany). Acetic acid, hydrochloric acid, and ammonium acetate were obtained from Panreac labs (Barcelona, Spain), and the solvents methanol and acetonitrile (LC-MS grade) were supplied by JT-Baker (Philipsburg, NJ, USA). Deionized water was purified using a Milli-Q system (Millipore, Bedford, MA, USA).
Trypsin-EDTA, Eagle’s minimum essential medium (EMEM), L-glutamine, foetal bovine serum (FBS), penicillin/streptomycin, and essential amino acids were purchased from ThermoFisher Scientific (Madrid, Spain). The flat bottom 96-well plates were obtained from Corning (New York, NY, USA). Trypan Blue was obtained from Sigma-Aldrich (St. Louis, MO, USA).

A reverse phase C18 column (Phenomonex, SphereClone 5µ ODS(2), 150 mm × 4.6 mm) was equilibrated for 30 min with a mobile phase which consisted of 100% diH₂O. Flow rate was set to 1 ml/min and samples separated according to programme for desulfoglucosinolates detailed in Table 3. Mobile phase solutions were degassed for 30 min in a sonicator (Decon, Sussex England).Table 3

Mobile phase conditions for separation of desulfoglucosinolates

Time	% Solution A	% Solution B	Transition
0	100	0
30	0	100	Linear gradient
35	0	100
40	100	0	Linear gradient
50	100	0

Solution A: 100% diH₂O

Solution B: 70:30, diH₂O:acetonitrile

Desulfoglucosinolates were quantified using 229 nm wavelength within the UV spectrum. The HPLC PDA detector allowed a full spectrum analysis from 180 to 800 nm, allowing comparative UV–visible spectra analysis, which aided in identifying unknown glucosinolates. Through standard injections and HPLC–MS identification we were able to confirm the id’s of these reported glucosinolates. Desulfated purified standards: sinigrin (sigma aldrich), glucotropaeolin, glucoraphenin, glucoraphanin, glucerucin, glucobrassicin, gluconasturtiin, sinalbin, progoitrin and glucoiberin (phytoplan).