Akta purifier upc 100

The ACE inhibitory activity of the different fractions obtained in the previous step was compared, and the most active fraction was separated and purified with an AKTA-FPLC system (AKTA Purifier UPC 100, GE Healthcare, Uppsala, Sweden). The sample solution (0.1 mg/mL) was filtered through a 0.45 μm microporous membrane and 500 µL was injected into the AKTA-FPLC system equipped with a Superose^® 12 10/300 GL agarose gel column (10 mm × 300 mm, 10 μm). Elution was carried out at 0.5 mL/min with ultrapure water, fractions were detected at 280 nm, and collected automatically (3.5 mL/tube). The fractions corresponding to each absorption peak were pooled and freeze-dried, and their ACE inhibitory activity was determined.

The flavonol glycoside-enriched fraction (uploading volume of 2.5 mL, 45% methanol fraction by Method B) was collected and concentrated by a rotary evaporator. The concentrate was reconstituted with 4 mL water and then filtered through a 0.22 µm membrane before injection into the preparative HPLC AKTA purifier UPC100 (GE Healthcare Bio-Sciences AB, Sweden). The preparative HPLC conditions were: YMC-Pack ODS-A column (5 µm, 250 × 10 mm), injection volume 0.5 mL, mobile phase A: acetonitrile/acetic acid/water = 3.0 : 0.5 : 96.5 (v/v/v), mobile phase B: water/acetonitrile/acetic acid = 30.0 : 0.5 : 69.5 (v/v/v), gradient elution: maintaining 55% A and 45% B during the first 35 min, and then linearly increasing to 35% A and 65% B till 55 min, and increasing to 100% B till 70 min, flow rate 2 mL/min. The fractions corresponding to Peaks 1–7 were collected for chemical analyses.

LJSF4 was prepared according to the method described in our previous study [25 (link)]. In brief, the defatted sample of S. japonica was extracted by triple volume of distilled water at 120 °C for 2 h, and then the supernatant was collected. Then, via adding an equal volume of 2% CaCl₂ solution in order to remove the alginate in the supernatant, and the resulting supernatant was finally lyophilized to gain crude fucoidan of LJS. The crude LJS was further purified by an anion exchange chromatography (AKTA Purifier UPC100, GE healthcare, Pittsburgh, PA, USA) to obtain the four fractions of LJSF1, LJSF2, LJSF3, LJSF4. The sulfate content and total sugar of each fraction were determined according to our published work [25 (link)]. LJSF4 was collected for further assays in this work.