Iridoid glycosides

To determine iridoid glycoside levels in P. lanceolata, plant samples were freeze-dried for 3 days under vacuum (− 55 °C collector temperature; Labconco Free Zone 12 L Freeze Dry System, USA), finely ground and weighed. Twenty-five mg of each sample was extracted overnight in 10 ml, at room temperature in 70% methanol (LichroSolv, VWR) using a horizontal shaker, then filtered and diluted ten times with ultrapure water. The concentrations of the IGs (aucubin and catalpol, Sigma-Aldrich) were analyzed using high-performance liquid chromatography (HPLC, Bioinert 1260 Infinity, Agilent) with electro chemical detection (ECD, Decade elite ECD, Antec). For HPLC quantification, five microliters of filtered extracts and standards was analyzed at 20 °C with a Dionex™ Guard column CarboPac PA1 2 × 50 mm, Main column CarboPac PA1 2 × 250 (Thermo Fisher Scientific). The isocratic mobile phase contained 100% 0.1 M NaOH at a flow rate of 0.25 ml/min, runtime 35 min. Retention time (RT) was 3 and 5 min for aucubin and catalpol, respectively. The standard concentration range was 0.125–2.5 ppm.