In vitro Effects of Saponins on Lysozyme Activity of Human Cells 343 Primulic acid at concentrations higher than 12.5µg/ml as well as Hydrocotyle vulgaris saponins at concentrations equal to or higher than 6.25µg/ml were toxic to the epithelial cells with distinct inhibition of lysozyme activity of cells. Concentration of 3.125µg/ml of these two saponins, which was non-toxic to cells, caused slight decreases in lysozyme activity of cell cultures after 24 hours incubation (Tab. 2). Tab. 2. Percent change (to control) in the total and secreted lysozyme activity of 0.5×10+6 cells/ml HT-29 cell cultures treated with various saponins for 24 hours. Conc. Secreted lys.a Total lys.a Cell viability Saponin (µg/ml) (%b) (%b) (%c) 3.125 −7 ±5 % 2 ±3 % 92 ±5 % Primulic acid 12.5 −16 ±3 %* 0 ±2 % 89 ±10 % 25 −71 ±2 %* −67 ±1 %* 45 ±6 %* Hydrocotyle vulg. 3.125 −11 ±3 %* −5 ±4 % 92 ±4 % sap. 6.25 −33 ±2 %* −25 ±3 %* 79 ±6 %* 12.5 −80 ±3 %* −79 ±2 %* 43 ±8 %* Aescin 25 −10 ±6 %* −4 ±5 % 90 ±4 % 50 −30 ±1 %* −21 ±2 %* 80 ±3 %* Saponinum album 12.5 −7 ±18 % −8 ±8 % 93 ±7 % 50 −59 ±1 %* −53 ±2 %* 47 ±8 %* Quillaja sap. 3.125 −6 ±1 % 1 ±3 % 95 ±3 % 50 −27 ±1 %* −17 ±2 %* 90 ±7 % a All points were measured in triplicate using sensitive fluorescence-based lysozyme activity assay, and each value is representative of average of at least six separate experiments (±SD); b Control cultures without saponin addition were considered to have 100% lysozyme activity; c Cell viability of saponin-treated cultures was calculated by comparison with the control cultures for the same incubation period. * Values with asterisk are significantly different (P<0.05, Mann-Whitney U test) from values of control cultures. Furthermore, aescin and saponinum album (from Gypsophila paniculata) at 50µg/ml exhibited toxic effects to the cells after 24 hours treatment, which led to significant decreases of total and released lysozyme activity. These two saponins at lower non-toxic concentrations resulted in slight decreases of lysozyme activity (Tab. 2). Quillaja saponaria saponins at non-toxic concentration of 50µg/ml caused significant decreases of 17% and 27% of both total and secreted lysozyme activity respectively after 24 hours incubation with epithelial cells (Tab. 2). Lower concentrations of Quillaja saponins had no effects on lysozyme activity. Another group of the selected saponins showed no significant influences on the production and release of lysozyme activity of the epithelial cells after 24 hours exposure regardless of the used non-toxic doses, including hederacosid C, Helianthus annuus saponins, ginsenosid-Rd and glycyrrhizinic acid. Sci Pharm. 2011; 79: 337–349