Culture flasks

Enisamium iodide and N-methyl-4-N-ethanolpyridinium iodide (internal standard for LC-MS/MS) were provided by Farmak JSC.
Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Pan Biotech (Aidenbach, Germany). [³H]-propranolol, rhodamine 123, Krebs-Ringer buffer (KRB), cyclosporine A and [³H]-digoxin from Sigma-Aldrich. Trypsin/ethylenediaminetetraacetic acid (EDTA) solution and sodium dodecyl sulfate from Merck (Darmstadt, Germany). Fluorescein was purchased from VWR International (Darmstadt, Germany). Culture flasks were obtained from VWR International and Transwell filter inserts from Corning Life Sciences (Lowell, MA, USA).

After the cultured cells (HaCaT and BJ) had reached the desired confluence, the DMEM medium was aspirated in the culture flasks (VWR, Radnor, PE, USA) and the bottom-attached cells were washed twice with sterile PBS (phosphate buffered saline, Gibco). The cell layer was detached with trypsin/EDTA (Gibco) and then the cells were placed in fresh DMEM medium. The cells were then seeded in 96-well flat bottom plates (VWR, Radnor, PE, USA). After attaching HaCaT cells and fibroblasts to the bottom of the plates, the cells were treated with PRE, PGE, KTE, CTE and GGE extracts at concentrations of 50, 250 and 500 μg/mL. Cells were then cultured in an incubator for 24 h. The controls were cells (separately HaCaT and fibroblasts) grown in DMEM medium without the addition of test extracts.