340 R. Helal and M. F. Melzig: Ginsenosid-Rd and primulic acid induced slight but significant increases in the release of lysozyme activity when incubated with cells for one hour. Ginsenosid-Rd stimulated lysozyme activity release about 25% higher than the control cultures after one hour treatment with no toxic effect, while primulic acid stimulated lysozyme activity release 15% higher than control cultures. No increases in the stimulatory effects of ginsenosid-Rd or primulic acid on lysozyme activity secretion could be detected with the increasing saponin concentrations. Nevertheless, primulic acid at 50µg/ml illustrated slight toxic effects on THP-1 cells that were statistically non-significant after one hour treatment, while lower concentrations were non-toxic to cells. Another group of saponins showed no influences on lysozyme activity secreted in THP-1 cell cultures after one hour treatment, including hederacosid C, glycyrrhizin, saponins from Gypsophila paniculata (saponinum album) and from Helianthus annuus marginal flowers. Furthermore, no significant differences were found when these saponins were used at different end-concentrations up to 100µg/ml. Synthesis & secretion of lysozyme activity in cell cultures after long-term treatment with saponins Lysozyme activity levels in THP-1 cultures supplemented with different saponins were measured at various incubation time points of up to 48 hours. Tab. 1. Percent change (to control) in the total and secreted lysozyme activity of 1×10+6 cells/ml THP-1 cell cultures treated with various saponins for 24 and 48 hours. Saponin After 24h incubation Conc. Secreted Total Cell (µg/ml) lys.a lys.a viability (%b) (%b) (%c) 6.25 −20 ±7* −35 ±2* 89 ±8 25 −27 ±6* −38 ±4* 91 ±5 Quillaja sap. Aescin Hydrocotyle vulg. sap. 3.125 −7 ±16 −13 ±27 90 ±5 Saponinum album 12.5 −6 ±21 −15 ±22 90 ±6 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. −21 ±2* −26 ±3* 89 ±10 After 48h incubation Secreted Total Cell lys.a lys.a viability (%b) (%b) (%c) −21 ±11* −12 ±4* 88 ±4 −16 ±4* −22 ±5* 90 ±8 −23 ±8* −25 ±2* 87 ±12 Contrary to the tendency towards increased lysozyme secretion observed with the short- term treatment with saponins, THP-1 cells treated with saponins for longer incubation periods up to 48 hours showed tendency to decrease in the secretion and production of lysozyme activity in the examined cultures. Sci Pharm. 2011; 79: 337–349