GC/MS analyses were performed on a Shimadzu QP5050A GC/MS system equipped with an AOC-20i auto-injector. A J&W Scientific DB-5MS fused capillary column (30 m × 0.25 mm × 0.25 μm film thickness; Agilent, Santa Clara, CA) was used as the stationary phase. MS data were taken at 70 eV with a scan interval of 0.5 s from m/z 40 to 500. All other conditions were similar to the GC analysis. Essential oil components were identified by comparing the retention times of the GC peaks with standard compounds run under identical conditions and by comparison of retention indices (Van Den Dool & Kratz, 1963) and mass spectra (Adams, 2007) with those

found in the literature, and by comparison of mass spectra with those stored in the NIST 107 LGK974 and NIST 21, and Wiley 229 libraries. Tumour cell growth was determined by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan product, as described by Mossman (1983). For all experiments, cells were seeded in 96-well plates (0.7 × 105 cells/ml for adherent cells or 0.3 × 106 cells/ml for suspended cells in 100 μl of medium). After 24 h, the drugs (0.78–50 μg/ml) were dissolved in pure DMSO and added to IDH inhibitor each well using HTS – High-Throughput Screening (Biomek 3000, Beckman Coulter Inc., Fullerton, CA). Then, the

cells were incubated for 72 h. Doxorubicin (purity > 98%; Sigma Chemical Co., St. Louis, MO) was used as positive control. At the end of incubation, the plates were centrifuged, and the medium was replaced by fresh medium (150 μl) containing 0.5 mg/ml MTT. Three hours later, the formazan product was dissolved

in 150 μl DMSO, and absorbance was measured using a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter Inc.). The drug effects were expressed as the percentage of control absorbance of reduced dye at 595 nm. The in vivo antitumour effect Cyclin-dependent kinase 3 was evaluated using Sarcoma 180 ascites tumour cells, following protocols previously described ( Bezerra et al., 2006, Bezerra et al., 2008 and Britto et al., 2012). Ten-day-old Sarcoma 180 ascites tumour cells (2 × 106 cells per 500 μl) were implanted subcutaneously into the left hind groin of mice. The essential oil was dissolved in 5% DMSO and given to mice intraperitoneally once a day for 7 consecutive days. At the beginning of the experiment, the mice were divided into four groups of 8–15 animals as follows: Group 1: animals treated by injection of vehicle 5% DMSO (n = 15); Group 2: animals treated by injection of 5-fluorouracil (5-FU, purity > 99%; Sigma Chemical Co.) (25 mg/kg/day) (n = 9); Group 3: animals treated by injection of the essential oil (50 mg/kg/day) (n = 8); Group 4: animals treated by injection of the essential oil (100 mg/kg/day) (n = 8). The treatments were started one day after tumour injection. The dosages were determined based on previous articles.