Deae sephadex a 25 anion exchange resin

Flower, leaf and stem, and root were ground to a fine powder. Samples of ca. 100 mg were extracted for 5 min at 80 °C in 2 × 1 mL MetOH/H₂O (70:30 v/v) to inactivate the endogenous myrosinase using a GLH 850 homogenizer (OMNI International, Kennesaw, GA, USA) and then centrifuged for 10 min. Supernatants were combined to reach a final volume of 2 mL. Each extract (1 mL) was loaded onto a mini-column filled with 0.6 mL of DEAE-Sephadex A-25 anion-exchange resin (GE Healthcare, Pittsburgh, PA, USA) conditioned with 25 mM acetate buffer (pH 5.6). After washing the columns with 2 mL of 70% MetOH for the removal of nonpolar compounds and 1 mL of ultrapure water, optimal conditions for desulfation were created by adding 2 mL of buffer solution. In addition, 20 μL (0.35 U/mL) of purified sulfatase [26 (link)] was loaded onto the mini-columns as well as 50 μL of buffer in order to distribute the sulfatase equally and were left to stand overnight at 30 °C. The DS-GSLs were then eluted with 1.5 mL of ultra-pure H₂O and the samples were stored at −20 °C until further analysis.

GSLs were extracted as previously reported [26 (link),28 (link)]. The plant material was divided into root, stem, leaf, and flower for Sisymbrium officinale and root, stem, and siliquae for S. orientale. The samples were freeze-dried using FreeZone 2.5 L freeze-dryer (Labconco, Kansas City, MO, USA) and ground to a fine powder, from which 100 mg were extracted for 5 min at 80 °C in 2 × 1 mL MeOH/H₂O (70:30 v/v) to inactivate the endogenous myrosinase. DEAE-Sephadex A-25 anion-exchange resin (10 g, GE Healthcare) was mixed with 125 mL of ultrapure water, and the resulting mixture was stored in a refrigerator (4 °C). Each extract (1 mL) was loaded onto a mini-column filled with 0.5 mL of DEAE-Sephadex A-25 anion-exchange resin solution (1 cm height × 0.5 cm diameter) and conditioned with 25 mM acetate buffer (pH 5.6). After washing the column with 70% MeOH and 1 mL of ultrapure water, the optimal conditions for desulfation were set by adding a buffer solution. Each mini-column was loaded with 20 μL (0.35 U/mL) of purified sulfatase and left to stand for 18 h at room temperature. The desulfoGSLs were then eluted with 1.5 mL of ultra-pure H₂O, lyophilized and diluted to 1 mL. The samples were stored at −20 °C until further analysis by HPLC-DAD-MS/MS.

GSLs were extracted as previously reported [31 (link),32 (link)]. The dried plant parts of Rab sample (aerial part) and Split sample (flower, leaf, stem, siliquae, root) were ground to a fine powder, from which 100 mg were extracted for 5 min at 80 °C in 2 × 1 mL MeOH/H₂O (70:30 v/v) to inactivate the endogenous myrosinase. Each extract (1 mL) was loaded onto a mini-column filled with 0.5 mL of DEAE-Sephadex A-25 anion-exchange resin (GE Healthcare, Chicago, IL, USA) conditioned with 25 mM acetate buffer (pH 5.6). After washing the column with 70% MeOH and 1 mL of ultrapure water, optimal conditions for desulfation were set by adding buffer solution. Each mini-column was loaded with 20 μL (0.35 U/mL) of purified sulfatase and left to stand 18h at room temperature. The desulfo-GSLs were then eluted with 1.5 mL of ultra-pure H₂O, lyophilized and diluted to the 1 mL. The samples were stored at –20 °C until further analysis by UHPLC-DAD-MS/MS.