Eurospher 100 5

Microdialysis samples were measured with a Knauer high-performance liquid chromatograph (HPLC) with ultraviolet (UV) detection and a Smart line pump (1000). The β-EP and MENK separation were carried out isocratically with a Knauer reverse phase C18 column (vertex plus 250 × 4.6 mm Eurospher100-5) with guard cartridge and a Knauer CTO-6A column oven. The system was used at oven temperature (40 °C) with detection at 210 nm. The flow rate was set at 0.2 mL/min to elute samples of the column. Solvents were degassed under vacuum. The buffer was KH₂PO₄ (68 mL) and acetonitrile (32 mL) 0.1 M (pH 2.3). The mobile phase was acetonitrile/buffer in the first 4 min, acetonitrile/water from 4 to 6 min, and acetonitrile/buffer from 6 to 12 min (temperature 40 °C). Plotting of graphs and data analyses were performed with Autochro data module software. The concentrations of each sample were calculated by interpolating the areas obtained after fraction injection with those obtained from three known standard concentrations (met-enk from Abcam and β-end from Sigma-Aldrich, dissolved in normal saline) injected regularly into the HPLC to monitor the linearity of the system. Integration of chromatographic data was performed with Perkin-Elmer 1020 software [41 (link)].

A Waters liquid chromatography device coupled with a Photodiode Array Detector (PDA) and C₁₈ column (Knauer, 25 cm × 4.6 mm Eurospher 100-5) was used for phytochemical quantification of the plant materials (the aerial and underground parts) according to Esmaeili et al.^{27 (link)}.