All the organic solvents were obtained from Carlo ERBA (Paris, France). L-glutamine was purchased from GIBCO BRL Life technologies (Grand Island, NY, USA). The N-(1-naphtyl) ethlenediaminedihydrochloride (EDTA) was purchased from Sigma–Aldrich (Steinheim, Germany). RPMI-1640, foetal bovine serum and gentamicin were bought from GIBCO BRL Life technologies (Grand Island, NY, USA) Folin–Ciocalteu reagent and 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP) were purchased from Wako Chemicals USA, Inc. (Richmond, VA). 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Dimethyl sulfoxide and acetic acid were obtained from Fisher Scientific (Pittsburgh, PA). Sodium carbonate, acetone, and methanol were obtained from Mallinckrodt Baker, Inc. (Phillipsburg, NJ).
Nitraria retusa was collected in December 2006 from the saline soils at Sahline, a region in the center of Tunisia. Identification was carried out by Pr. M. Cheieb (Department of Botany, Faculty of Sciences, University of Sfax, Tunisia) according to the Flora of Tunisia [11, 12]. A voucher specimen (N.r-12.06) was kept at our laboratory for future reference. The leaves were shade-dried, powdered and stored in a tightly-closed container for further use.
Preparation of ethyl acetate extracts from Nitraria retusa leaves
Three hundred and fifty grams of powder, from dried leaves, were sequentially extracted in a Soxhlet apparatus (6 h) (AM Glassware, Aberdeen, Scotland, United Kingdom) with hexane, chloroform, ethyl acetate and methanol solvents. We obtained the corresponding extract for each solvent. They were concentrated to dryness and kept at 4°C.
Fractionation methods and structural identification of the purified compound
The ethyl acetate extract was fractionated by vacuum liquid chromatography (VLC) on a silica gel column and rechromatographed over RP18 column using medium liquid pression column (MLPC). Four sub-fractions were gathered, their purity was verified by thin layer chromatography, then identified by comparison of their Nuclear Magnetic Resonance (NMR) data to the literature to enable the identification of compound 1.
Cell culture and assay for cytotoxic activity
Human chronic myelogenous leukaemia CML cell line K562 was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) foetal calf serum, 0.1 mg/ml gentamicin and 2 mM L-glutamine as complete growth medium, and were incubated at 37°C in an incubator with 5% CO2, in humidified atmosphere. Every 2 days, cells were split for subculture with fresh medium.
The cytotoxicity of N. retusa extract and I3- O-Rob against the K562 cells were estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, previousely described by Boubaker et al., .
Evaluation of the antioxidant capacity of EA extract and I3-O-Rob against lipid peroxidation provoked by H2O2, using the thiobarbituric acid reactive substance (TBARS) assay:
The method known as thiobarbituric acid reactive species (TBARS) assay concerns the spectrophotometric measurement of the pink pigment produced through the reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA) and other secondary lipid peroxidation products. TBARS were determined according to the assay described by Ohkawa . The cells (3.5 x 107 cells/ml) were exposed to various concentrations of each sample (250, 500 and 1000 μg/ml of I3-O-Rob and 200, 400 and 800 μg/ml of EA extract) in the incubation medium for 2 h, followed by incubation with 50 μM H2O2 for 2 h. The doses ranges of the different tested compounds were chosen on basis of their cytotoxic activity. The cells were washed with PBS, pelleted and homogenized in 1.15% KCl. The samples were combined with 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid and 1.5 ml of 0.8% thiobarbituric acid. The mixture was brought to a final volume of 4 ml with distilled water and heated to 95°C for 120 min. After cooling for 10 min on ice, 5.0 ml of a mixture of n-butanol and pyridine (15:1 v/v) were added to each sample, and the mixture was shaken vigorously. After centrifugation at 825 g for 10 min, the supernatant fraction was isolated and the absorbance was measured at 532 nm. The lipid peroxidation effect was expressed as equivalent of MDA. Data were reported as mean ± SD for triplicate determinations.
Cellular antioxidant activity (CAA) assay
Human myelogenous erythroleukaemia K562 cells were seeded at a density of 6 × 104/well on a 96-well microplate. Twenty-four hours after seeding, the growth medium was removed and the wells were washed with PBS. Triplicate wells were treated for 1 h with 100 μL of extracts plus 25 μM DCFH-DA dissolved in treatment medium. The wells were washed with 100 μL of PBS, then 600 μM ABAP solution was applied to the cells in 100 μL of PBS, and the 96-well microplate was placed into a Fluoroskan Ascent FL plate-reader (ThermoLabsystems, Franklin, MA) at 37°C. Emission at 538 nm was measured with excitation at 485 nm, every 5 min for 1 h. Each plate included triplicate control and blank wells: The control wells contained cells treated with DCFH-DA and oxidant ABAP; the blank wells contained cells treated with PBS without oxidant [14–16].
Quantification of CAA
After blank subtraction from the fluorescence readings, the area under the curve of fluorescence versus time was integrated to calculate the CAA value at each concentration of the samples as follows:
where ∫SA is the integrated area under the sample fluorescence versus time curve and ∫CA is the integrated area from the control curve. The median effective dose (IC50) was determined for the pure phytochemical compounds and the leaf extract from the median effect plot of log (f a/ f u) versus log (dose), where f a is the fraction affected and f u is the fraction unaffected by the treatment. To quantify the intraexperimental variation, the IC50 values were stated as mean SD for triplicate sets of data obtained from the same experiment [14–16].
The Comet assay with human lymphocytes was used to detect DNA damage. Before each experiment, frosted microscope slides were precoated with 2 layers (100 μl) of normal agarose (1% in milli-Q water) and left at room temperature to allow agarose to dry. The cells were treated during 24 h with different concentrations of the tested samples. The treated cells were stressed with 75 μM H2O2 for 2 h. The cell dilution (5 × 105 cells in 60 μl) was mixed with an equal volume of low-melting point agarose (1.2% in PBS). This agarose cell suspension (120 μl) was spread onto each precoated slide and covered with a cover slip. After 10 min on ice, the cover slip was gently removed, and the slides were placed in a tank filled with the lysate buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris–HCl, 1% sodium sarcosinate pH 10, 1% of Triton X-100, and 10% DMSO). They were immersed for 1 h in this buffer (4°C, in the dark). The slides were then transferred into the electrophoresis buffer (NaOH 10 N, EDTA 200 mmol/l, pH 13 in deionized water) for 20 min at room temperature in darkness. Electrophoresis was carried out for 15 min at 25 V, 300 mA. Finally, the slides were gently rinsed with neutralization solution (0.4 M Tris–HCl, pH 7.5) 3 times for 5 min each. Staining of DNA was accomplished using 50 μl of ethidium bromide solution (20 μg/ml in PBS) per slide . The slides were examined using an epifluorescence microscope (Zeiss Axioskop 20; Carl Zeiss, Microscope Division, Oberkochen, Germany).
Quantification of the comet assay
A total of 100 comets on each scored slide for each sample concentration were visually-scored according to the relative intensity of fluorescence in the tail and classified as belonging to one of five classes. We utilised three slides for each extract concentration, and the experiments were repeated three times. Each comet class was given a value of 0, 1, 2, 3, or 4 (from undamaged, 0 to maximally damaged, 4) as described previously by Collins et al. . The total score of DNA damage was calculated by the following equation:
The inhibition percentage of Tail DNA (%) was calculated relative to DNA damage in the control group cells treated with H2O2 only by the following formula:
Data were collected and expressed as the mean ± standard deviation of three independent experiments and analysed for statistical significance from positive control. The data were tested for statistical differences by student test, differences were considered to be significant when p < 0.05.