2.2.3. Chromatography

Chromatographic analysis was performed by using high-performance liquid chromatography (HPLC) (Perkin Elmer, Norwalk, CT, USA) Series 200 pump equipped with an LC-235C Diode Array Detector (DAD), auto-sampler, and column oven. Chromatographic data were processed by using a Turbochrom Workstation software, version 6.1.2 (Perkin Elmer, Norwalk, CT, USA). Separation and determinations were accomplished on a 5 μm Hypersil ODS RP-18 column (250 × 4.6 mm, Supelco, Bellefonte, PA, USA) fitted with a guard column (Hypersil ODS RP-18, 5 μm particles, 10 × 4.6 mm, Supelco, Bellefonte, PA, USA). All the samples were filtered, through 0.45 μm membrane filter, and degassed by an ultrasonic bath before the injection. The procedure was performed, as previously described [17].

#### 2.2.4. Determination of Antioxidant Activity in an In Vitro Cellular Free System

The stable DPPH radical was used for the determination of free radical-scavenging activity of the extracts [29]. Because of its odd electron, the DPPH radical gives a strong absorption band at 517 nm in visible spectroscopy (deep violet color). As this electron becomes paired off in the presence of a free radical scavenger, the absorption vanishes, and the resulting decolorizing is stoichiometric with respect to the number of electrons taken up. The reaction mixture contained 86 μM DPPH radical and different concentrations of each extracts (0.025-0.5-0.1-0.2-0.4 mg/mL) in 1 mL of ethanol. After 10 min at room temperature, the absorbance at 517 nm was recorded [30]. Trolox (30 μM), water-soluble derivative of vitamin E, was used as a standard. The assay was performed in triplicates.

#### 2.2.5. Determination of Antioxidant Activity within an In Vitro Cellular System

Primary cultures of astrocytes were prepared from new-born albino rat brains (from 1-day-old to 2-day-old Wistar strain rats) as described [31]. Cerebral tissues, after dissection and careful removal of the meninges, were mechanically dissociated through 82 μm pore sterile mesh (Nitex, Darmastadt, Germany). Isolated cells were suspended in DMEM, supplemented with 20% (*v*/*v*) FBS, 2 mM glutamine, streptomycin (50 mg/mL), and penicillin (50 U/mL), and plated at a density of 3 × 10<sup>6</sup> cells/100 mm dishes and of 0.5 × 10<sup>5</sup> cells/chamber of multi-chambered slides. Cells were maintained at 37 ◦C in a 5% CO2 and 95% air humidified atmosphere for two weeks. The medium was exchanged every three days. The low initial plating density of dissociated cells was meant to favor the growth of astrocytes with only a very little oligodendroglial and microglial cells contamination. Astroglial cell cultures were characterized at 14 days in vitro (DIV), when it is confluent, by immunofluorescence staining with GFAP. All experiments conformed to the guidelines of the local Ethical Committee (University of Catania, Italy), and were carried out in accordance with the EC Directive 86/609/EEC for animal experiments.

Astrocytes at 14 DIV were treated with glutamate (500 μM) for 24 h, as previously described [32]. Those cultures were treated with different concentrations of SN1 and SN2 (0.5 and 1 mg/mL), which were added 20 min before glutamate exposure. Four replicates were carried out for each sample. In a subset of experiments, to assess the inhibition of glutamate effects, the astroglial cell cultures were incubated 20 min prior to glutamate exposure, with GYKI 52466 (100 μM), the specific AMPA/KA receptor antagonist.

Astroglial cell survival analysis was performed by the MTT reduction assay, which evaluated mitochondrial dehydrogenase activity, as previous reported [33,34]. Astrocytes were set up 0.5 × 10<sup>5</sup> cells per well of a 96-multiwell, flat-bottomed, 200-μL micro plate. They were maintained at 37 ◦C in a humidified 5% CO2 and 95% air mixture [33]. At the end of treatment time, 20 μL of 0.5% MTT in (pH 7.4) PBS were added to each micro-well. After 1 h of incubation with the reagent, the supernatant was removed and replaced with of DMSO (200 μL). The optical density of each well was measured with a micro-plate spectrophotometer reader (Titertek Multiskan, Flow Laboratories, Helsinki, Finland) at l = 570 nm.

## 2.2.6. Glutathione Measurement

Astroglial cell cultures were scraped off and lysed in 50 μM sodium phosphate buffer (pH 7.4). The Bradford assay determined the protein concentration in cell extracts [32]. Then, a Hitachi U-2000 spectrophotometer (Hitachi, Tokyo, Japan) chemically determined the total glutathione intracellular content (GSH + GSSG), as described by Chen YH et al. [35].

#### 2.2.7. ROS Levels Determination

Reactive species determination was performed by using DCFH-DA as a fluorescent probe. Furthermore, 100 μM of DCHF-DA was dissolved in 100% methanol, added to the cellular medium, and the cells were incubated at 37 ◦C for 30 min. Under these conditions, the acetate group was not hydrolyzed [12]. After incubation, astroglial cell cultures were lysed and centrifuged at 10,000× *g* for 10 min. The fluorescence corresponding to the radical oxidized species 2-,7--dichlorofluorescein (DCF) was monitored by measuring the excitation (λ = 488 nm) and emission (λ = 525 nm), using an F-2000 spectrofluorometer (Hitachi).

Values are expressed as a percentage of fluorescence intensity per mg protein *versus* control (% I.F/mg prot vs. control). Protein concentration was measured, according to the Bradford assay applied by Li Volti et al [32].
