Samples of OLSx 1–6 were analysed using a Q-Trap mass spectrometer (Applied Biosystems) with the direct infusion of the sample solutions into the electrospray ionisation source operating in the buy ABT-263 negative ion mode. Capillary and cone voltages were set to −4500 V and −50 V, respectively, with a de-solvation temperature of 100 °C. OLSx 3–6 were introduced into an HPLC (Agillent) with a μBondapak C18 analytical column (Waters, 3,9 × 300 mm, 10 μm) and detected in a Q-Trap mass analyser. ESI(−)–MS was carried out with capillary and cone voltages set to −4500 and −50 V, respectively, and a de-solvation temperature of 300 °C. A binary mobile phase of acetonitrile and 1% of

formic acid was employed. A linear gradient was performed starting from 30% of acetonitrile to 100% acetonitrile, in 30 min, and an elution flow rate of 1 ml/min. Tandem mass spectra were acquired using a hybrid high-resolution and high-accuracy (5 ppm) Micromass Q-TOF mass spectrometer (Waters) and via collision-induced dissociation at ca. 15 V. Capillary and cone voltages were set to ±3000 and ±40 V, respectively, for the negative or positive mode of ionisation. INK1197 manufacturer The de-solvation temperature was 100 °C; nitrogen and argon were used as de-solvation or collision

gas, respectively. The cytotoxicity of propolis extracts and fractions from ODEP was evaluated against four human tumour cell lines: HL-60 (leukemia), HCT-8 (colon), MDA/MB-435 (breast) and SF-295 (brain) obtained from Amisulpride the National Cancer Institute (Bethesda, MD, USA). The general viability of cultured cells was determined by the reduction of the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a blue formazan product, as previously described by Mosmann (1983). The tumour cells were maintained in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin at 37 °C with 5% CO2. For all experiments

cells were seeded at 0.3 × 106 cells/ml (HL-60, MDA/MB-435 e SF-295) and 0.7 × 105 cells/ml (HCT-8), and incubated during 72 h with propolis extracts (0.001–50 μg/ml ODEP and EEP70) and fractions (0.001–25 μg/ml), under the conditions described above. After centrifugation and solution removing, MTT solution was added and the plates were incubated, centrifuged, and the solids dissolved in pure and sterile DMSO. The absorbance was measured in a plate spectrophotometer DTX-800 (Beckman Coulter) at 595 nm. Doxorubicin (Sigma) was used as a positive control. A total of 80 Swiss mice (male, 25–30 g), obtained from the central animal house of Federal University of Ceará, Brazil, were used. The animals were housed in cages with free access to food and water (conforming to a well-defined rodent diet). All animals were kept under a 12:12 h light–dark cycle (lights on at 6:00 a.m.).