346 Kits Saponins R. Helal and M. F. Melzig: EnzChek® Lysozyme Assay Kit was purchased from Molecular Probes™ (Invitrogen Detection Technologies, USA). DQ lysozyme substrate (Micrococcus lysodeikticus) stock suspension (1.0mg/ml) and 1000units/ml lysozyme stock solution were prepared according to the manufacturer. Ginsenosid-Rd, glycyrrhizinic acid, hederacosid C and saponins of Quillaja saponaria bark were purchased from Carl Roth GmbH, Germany. Saponinum album (isolated from Gypsophila paniculata L.) was purchased from Merck, Germany. Primulic acid (isolated from Primula officinalis L.) was purchased from Fluka AG, Germany. Saponins of Helianthus annuus L. were isolated from air-dried marginal flowers by Prof. Zieschang, Humboldt-Universität Berlin, Germany. Aescin was prepared according to Ph. Eur. standards. Saponins from Hydrocotyle vulgaris L. were isolated and identified by Prof. Heller, Humboldt-Universität Berlin, Germany. Each examined saponin was dissolved in PBS in a 1mg/ml stock solution, and then diluted with culture medium to the final concentrations. Cell culture The human leukemia nonadherent cell line THP-1 and human colon epithelial cell line HT-29 were maintained in RPMI-1640 medium without phenol red, supplemented with 10% fetal bovine serum and L-glutamine. Incubation was carried out at 37°C in a humidified atmosphere of 5% CO2/95% air. Cell cultures were passaged every 3–4 days. Passages from 5 to 30 were used for the experiments. Cell counts and viability were determined with Casy® cell counter (Schärfe System GmbH). Incubation of cell cultures with saponins THP-1 and HT-29 cells were counted, viability determined and pelleted by centrifugation. Cells were seeded into 24-well plates at an initial concentration of 1.0x10+6 cells/ml for monocytic cells and 0.5x10+6 cells/ml for epithelial cells. Incubation was carried out at 37°C in a humidified atmosphere of 5% CO2 for various incubation periods up to 72 hours in culture medium, or in preparations containing culture medium and one of the examined saponins in question. Cell cultures containing PBS (solvent of the examined saponin) served as the respective controls. Aprotinin was added to the incubation culture medium of THP-1 at a final concentration of 2µg/ml (0.3µM), in order to achieve best lysozyme determination results, as proved by previous experimental results [18]. With HT-29 cell line, aprotinin addition to culture medium was not necessary, because it had no enhancing effects on the lysozyme activity assay results (Data not shown). Determination of secreted lysozyme activity Supernatants of the examined and control cell cultures were collected at various time points according to the incubation period examined, and used to determine lysozyme activity released into culture medium. Sci Pharm. 2011; 79: 337–349