Deae sephadex a 25

Sinigrin hydrate, TBA hydrogen sulfate, sodium acetate, potassium sulfate, and DEAE-Sephadex A-25 were procured from Sigma-Aldrich (St. Louis, MO, USA). Enzyme sulfatase (Helix pomatia Type H-1), Dulbecco’s modified Eagle’s medium (DMEM), trypsin, ethylenediaminetetraacetic acid, penicillin-streptomycin solution, fetal bovine serum, trypan blue, MTT, and Annexin V-FITC apoptosis detection kit were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Caspase-3 activity assay kit was obtained from Elabscience (Houston, TX, USA). Human prostate cancer cell line (DU-145), human colon adenocarcinoma cell line (HCT-15), and human melanoma cell line (A-375) were generously provided by the National Centre for Cell Science (Pune, Maharashtra, India). All the cells were grown in DMEM culture media. HPLC grade methanol, water, and ACN were purchased from SD Fine-Chem (Mumbai, Maharashtra, India).

Samples were prepared as described by Hornbacher et al. [35 (link)]. Briefly, frozen plant materials were lyophilized in a freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) for 2 days and ground to a fine powder with a shaking ball mill (Retsch GmbH, Braunschweig, Germany). Approximately 50 mg dry plant tissue was extracted with 1 mL 80% methanol at room temperature for 10 min and then centrifuged at 13,000× g for 5 min. Before the centrifugation, samples were put on a shaker for 15 min after the first extraction and 30 min after the second extraction at room temperature (RT). The supernatants were pooled and loaded onto a column (polypropylene column, 1 mL) containing 2 mL of a 5% (w/v) suspension of DEAE Sephadex A25 (Sigma-Aldrich, Taufkirchen, Germany) in 0.5 M acetic acid (pH 5). Columns were washed five times with 2 mL H₂O and two times with 2 mL 0.02 M acetic acid (pH 5). For desulfation, 50 μL of sulfatase (Sigma-Aldrich, Taufkirchen, Germany) solution was added to 450 µL 0.02 M acetic acid (pH 5) and loaded onto the columns as well [36 (link)]. Desulfation took place for 24 h at RT. Afterwards desulfated GSLs were eluted three times with 2 mL HPLC H₂O (Sigma-Aldrich, Taufkirchen, Germany), dried overnight in a vacuum centrifuge and then dissolved in a total amount of 300 μL HPLC H₂O.

The GSLs extraction procedure was performed according to the method described by Bhandari et al. [26 (link)]. Briefly, freeze-dried and powdered cabbage samples (0.05 g) were extracted twice with 1 mL of boiling methanol (70%) for 20 min to deactivate the myrosinase. After centrifugation (12,000 × g, 10 min, 4 °C), the supernatant was transferred to a 2-mL tube. The supernatant from the two extractions was combined and considered as crude GSLs. The crude GSLs extract was added into a Mini Bio-Spin Chromatography Column (Bio-Rad Laboratories, Hercules, CA, USA), filled with 0.5 mL diethylaminoethyl (DEAE)-Sephadex A-25 (Sigma Aldrich, St. Louis, MO, USA), which had been activated with 0.1-M sodium acetate (pH 4.0). Then, 200 µL of purified aryl sulfatase (EC 3.1.6.1, type H-1 from H. pomatia; Sigma-Aldrich) was added, the column was capped from both sides and left for 18 h at room temperature for the desulfation of GSLs. The desulfo-GSLs were diluted with 3 × 0.5 mL distilled water, filtered through a 0.2-µm PVDF syringe filter and analyzed immediately using HPLC.