1100 series preparative hplc

Reversed-phase HPLC analysis of the refined water subfraction revealed the presence of multiple components, which necessitated further separation under preparative HPLC (Agilent 1100 Series Preparative HPLC, Santa clara, CA, US). Isolation of subfractions from the above fraction was carried out under conditions indicated in Table S1.
Purified subfractions/compounds were dried in vacuo at 40 °C, followed by freeze-drying for 24 h. The recovered compounds were stored at −15 °C until they were needed for further experiments. Bioactivities of each recovered subfraction (compound) were tested using either broth microdilution assay or disc diffusion assay in cases where only small amounts were recovered.

For the purification of the formed ZEN-metabolites an 1100 series preparative HPLC from Agilent Technologies equipped with a Gemini NX C18 column (150 mm × 21.2 mm; 5 µm; Phenomenex) was used. The eluents were water (eluent A) and methanol (eluent B) and the following gradient with a flow rate of 16 mL·min⁻¹ was applied: 40% B were held for 1 min and then the percentage of B was linearly increased to 100% up to 10 min. The column was flushed with 100% B for 3 min before switching back to the initial conditions within 0.1 min and column equilibration. The default injection volume was 900 µL and the peaks were collected by peak based fractionation (up and down slope 10 units·s⁻¹, max. peak duration 0.5 min) based on the UV signal at 270 nm between 4 and 10.5 min. ZEN-14,16-diG eluted at 4.7 min, ZEN-16-G at 6.1 min, ZEN-14-G at 7.5 min and ZEN at 9.7 min.