The roots of A. lappa (Asteraceae) were collected at CPQBA, University of Campinas (UNICAMP), experimental field (Paulínia, Brazil) in August 2007. Dr. Glyn Mara Figueira was responsible for identification of the plant species. A voucher specimen was deposited at UNICAMP Herbarium under number 146021.
Fresh milled roots (770 g) were extracted successively in a Soxhlet apparatus with dichloromethane, 95% ethanol and water (2:1 solvent/plant ratio), for 6 hours each solvent. The extracts were concentrated under vacuum (Buchi RE 215) until complete elimination of the organic solvent and subsequently freeze-dried for water elimination, providing dichloromethane (DHE), ethanolic (EHE) and aqueous hot extract (AHE).
Fresh milled roots (276 g) were successively extracted by dynamic maceration with dichloromethane, 95% ethanol and water (1:5 plant/solvent ratio, 3 times each solvent), at room temperature, in an oscillating agitator (FANEM). The extracts were concentrated under vacuum (Buchi RE 215) until complete elimination of the organic solvent and subsequently freeze-dried for water elimination, providing dichloromethane (DE), ethanolic (EE) and aqueous (AE) extracts.
Fresh milled roots (100 g) were extracted three times consecutively in Soxhlet extractor with water (1:5 plant/solvent ratio). The aqueous extract was freeze-dried, providing the total aqueous extract (TAE).
Fresh milled roots (594 g) were extracted three times with 70% ethanol (1:5 plant/solvent ratio) under reflux, for 6 hours. The filtrates obtained were combined and concentrated under vacuum. The remaining water was freeze-dried resulting in the hydroethanolic extract (HE).
High-resolution electrospray ionization mass spectrometry (HRESI-MS) of hydroethanolic extract
HRESI-MS was recorded on a Q-Tof Mass Spectrometer (Micromass - U.K.) using direct infusion of a 10 μL.min-1 MeOH + 0.1% formic acid solution and ionization by electrospray in the negative ion mode. Major operation conditions were as follows: capillary voltage of 3.5 kV, source temperature of 100°C, desolvation temperature of 100°C and cone voltage of 35 V.
2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
Microplate DPPH assay was performed as described by Brand-Williams et al. , modified by Brem et al. . Briefly, in a 96-well plate, successive sample dilutions (100 μL/well, 0.25, 2.5, 25 and 250 μg/mL), tested in triplicate, received DPPH solution (40 μM in methanol, 100 μL/well) and absorbance was measured at 550 nm with a microplate reader (VERSA Max, Molecular Devices). Results were determined every 5 min up to 150 min in order to evaluate the kinetic behavior of the reaction. The percentage of remaining DPPH was calculated as follows: % DPPH rem = 100 × ([DPPH] sample/[DPPH] blank). A calibrated Trolox standard curve was also made. The percentage of remaining DPPH against the standard concentration was then plotted in an exponential regression, to obtain the amount of antioxidant necessary to decrease the initial DPPH concentration by 50% (EC50). The time needed to reach the steady state for EC50 is defined as TEC50. The antiradical efficiency , was calculated as follows: AE = 1/(EC50 × TEC50).
Total phenolic content
The total phenolic content was performed as described by Prior et al. , with small modifications in order to use a microplate reader. Briefly, an aliquot (10 μL) of the sample (1 mg/mL) was diluted in distilled water (600 μL). Then, this solution was applied in a 96-well plate (150 μL per well), in triplicate, and received Folin-Ciocalteau solution (12.5 μL), sodium carbonate (37.5 μL, 1 M) and water (50 μL). After incubation at 37°C for 2 h, absorbance was measured at 725 nm with a microplate reader (VERSA Max, Molecular Devices). A calibrated gallic acid standard curve was made and results were expressed as mg equivalents in gallic acid per gram of sample.
In vitro antiproliferative activity assay
Human tumor cell lines UACC-62 (melanoma), MCF-7 (breast), NCI-ADR/RES (ovarian expressing phenotype multiple drug resistance), 786-0 (renal), NCI-H460 (lung, non-small cells), PC-3 (prostate), OVCAR-3 (ovarian), HT-29 (colon), K562 (leukemia) were kindly provided by Frederick Cancer Research & Development Center - National Cancer Institute - Frederick, MA, USA. Stock cultures were grown in 5 mL of RPMI 1640 (GIBCO BRL, Life Technologies) supplemented with 5% fetal bovine serum. Penicilin: streptomycin (1000 μg/mL:1000 UI/mL, 1 mL/L) were added to the experimental cultures. Cells in 96-well plates (100 μL cells/well) were exposed to each extract in DMSO (0.25, 2.5, 25 and 250 μg/mL) at 37°C, 5% of CO2 for 48 h. The final concentration of DMSO did not affect the cell viability. Then, a 50% trichloroacetic acid solution was added and after incubation (30 min at 4°C), washing and drying, cell proliferation was determined by spectrophotometric quantification (540 nm) of cellular protein content using sulforhodamine B assay. Using the concentration-response curve for each cell line the TGI (= concentration that produces total growth inhibition or a cytostatic effect) were determined through non-linear regression analysis using the software ORIGIN 7.5 (OriginLab Corporation) and corresponded to the test extract concentration necessary to inhibit proliferation of the cells.