330 M. Jenny et al.: maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (Biochrom, Berlin, Germany), 1% of 200 mmol/L glutamine (Serva, Heidelberg, Germany) and 0.1% of gentamicin (50 mg/ml, Bio-Whittaker, Walkersville, MD) in a humidified atmosphere containing 5% CO2 for 48h. This procedure was observed earlier to reveal best reproducible results when applied for testing of anti-inflammatory effects of compounds or drugs [39]. Average tryptophan content in the supplemented RPMI 1640 medium was 31.5 µmol/L. For each of the four experiments run in duplicates, PBMC were freshly prepared. Isolated PBMC were plated at a density of 1.5 x106 cells/ml in supplemented RPMI 1640, preincubated for 30 minutes with or without aqueous Crinum latifolium leave extracts and stimulated or not with 10 µg/ml PHA for 48h. Prostate carcinoma PC-3 cells were grown in Coon's F12 Modified Medium (Biochrom, Berlin, Germany) supplemented with 7% FCS, 4 mM L-glutamine and 50 µg/ml gentamycin. Benign prostatic hyperplasia (BPH-1)- and androgen-sensitive human prostate adenocarcinoma LNCaP cells were grown in RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 10% FCS, 4 mM L-glutamine and 50 µg/ml gentamycin. Cells were maintained in a humidified atmosphere containing 5% CO2. Oxygen radical absorbance capacity (ORAC) assay The capacity of the aqueous Crinum latifolium extract to scavenge peroxyl radicals was evaluated by the ORAC assay using the protocol of Ou. et al. [40]. Briefly, the final assay mixture (0.2 ml total volume) contained 6.3 x −8 M fluorescein (AnaSpec, USA) as a target of free radical attack and 1.9 x 10−2 M 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH, Wako Chemicals, Germany), which decomposes in aqueous solutions at 37°C and generates alkyl radicals (R´•) that are converted into the corresponding peroxyl radicals (R´OO•) in the presence of oxygen. 75 mM phosphate buffer served as a blank, and 6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, a synthetic water-soluble analogue of vitamin E, Sigma-Aldrich, Austria) was used as the control standard. The fluorescence of fluorescein was recorded at 37°C by a fluorometer (Fluoroscan Ascent, Labsystems; 485/535nm). During an assay period of 35 min the decrease of fluorescence was measured in minute intervals after the addition of AAPH. Final results were calculated using the differences between areas under the fluorescein decay curves (AUC) of blank and sample. The protective effect of a putative antioxidant was calculated by comparing the AUC of the sample with that of Trolox. The final ORAC values were expressed as µmol Trolox equivalents per gram of dried powder (µmol TE/g). Measurement of tryptophan, kynurenine and neopterin concentrations After incubation of cells for 48h, supernatants were harvested by centrifugation and tryptophan and kynurenine concentrations were determined by HPLC using 3-nitro-L- tyrosine as an internal standard [41]. To estimate IDO enzyme activity, kyn/trp was calculated and expressed as µmol kynurenine/mmol tryptophan. Neopterin concentrations were determined by an enzyme-linked immunosorbent assay (ELISA; BRAHMS, Hennigsdorf/Berlin, Germany) according to the manufacturer’s instructions with a detection limit of 2 nmol/L. Measurement of cell viability After incubation of PBMC with increasing concentrations of Crinum latifolium extract, the cells were either left untreated or stimulated with PHA [10 µg/ml] for 48 hours. Cell viability Sci Pharm. 2011; 79: 323–335