**3. Results**

#### *3.1. Analysis of SN Extracts*

The total phenolic contents was assayed by a modified Folin-Ciocalteu method using gallic acid, and is reported in Table 1, with the physical characteristics and the dry weight yields of SN1 and SN2.


**Table 1.** Extraction yields and concentration of total phenolic content of SN1 and SN2 extracts (*n* = 3).

The relative concentrations of phenolic acids and flavones in SN1 and SN2 were determined by HPLC analysis, according to Huang et al. [29]. The values were expressed in mg/g of dry extract. The results of the five phenolic acids determination (gallic, protocatechuic, chlorogenic, gentisic, and caffeic) and two flavones (luteolin and apigenin) in studied extracts are summarized in Table 2 while Figure 1 shows a representative chromatogram.

It was found that the major compound in both extracts was chlorogenic acid, whereas gentisic acid is the second. Luteolin is more abundant than apigenin, and less amounts of caffeic acid and protocatechuic acid were found. Gallic acid exists only in traces together with other unknown compounds.

**Table 2.** Contents of phenolic acid and flavones (mg/g of dry weight) in SN1 and SN2 leave extracts (*n* = 3).


**Figure 1.** Representative chromatogram of the dry extract reporting the retention time (RT) of gallic acid (1, 0.65 min), protocatechuic acid (2, 13.85 min), chlorogenic acid (3, 20.5 min), gentisic acid (4, 25.1 min), caffeic acid (5, 27.5 min), luteolin (6, 52.9 min), and apigenin (7, 70.95 min). Axis: x label minutes, y label absorbance unit.

#### *3.2. Antioxidant Activity in a Cellular Free System*

The free radical-scavenging activity of SN1 and SN2 extracts was tested by their ability to bleach the stable DPPH radical. Both extracts were able to quench the DPPH-radical at all the concentrations used (0.025-0.5-0.1-0.2-0.4 mg/mL) in a dose-dependent manner. SN1 showed a more potent capacity than SN2. In addition, their effect appeared similar to Trolox (30 μM), which is a soluble analogous of vitamin E used as a standard. This experiment demonstrated that SN1 and SN2 possess antioxidant properties (Figure 2).

**Figure 2.** Quenching of DPPH of SN1 and SN2 extracts at different concentrations (0.025-0.5-0.1-0.2-0.4 mg/mL), compared to Trolox (30 mM). Axis: x label: concentration, y label: quenching of DPPH expressed as a percentage. (\* *p* < 0.05 and \*\* *p* < 0.001).

#### *3.3. Antioxidant Activity in the Cellular System*

Primary rat astroglial cultures exposed to 500 μM glutamate for 24 h were used as an in vitro cellular model to assess the antioxidant effect of SN1 and SN2 extracts. Cells were characterized by immuno-fluorescent staining using GFAP as a marker [31].

The glutamate-evoked oxidative stress was evaluated by measuring the depletion of intracellular GSH levels (Figure 3) and ROS production (Figure 4). Glutamate (500 μM for 24 h) produced a significant decrease in the intracellular GSH levels and a significant increase of ROS levels, when compared to the untreated control ones. The pre-incubation of the cultures with SN1 and SN2 extracts (0.5 and 1 mg/mL) was able to restore, in a dose-dependent manner, GSH and ROS levels. In particular, 1 mg/mL of the extracts showed values similar to untreated control values.

**Figure 3.** GSH levels in primary rat astroglial cell cultures at 14 DIV: exposed to glutamate 500 μM for 24 h. Bar 1: control. Bar 2: cell culture exposed 500 μM. Bar 3: cell culture exposed 500 μM plus SN1 0.5 mg/mL. Bar 4: cell culture exposed 500 μM plus SN1 1 mg/mL. Bar 5: cell culture exposed 500 μM plus SN2 0.5 mg/mL. Bar 6: cell culture exposed 500 μM plus SN2 1 mg/mL. Four replicates were carried out for each sample. (\* *p* < 0.05 and \*\* *p* < 0.001). Axis: x label: concentration, y label: nmoli of GSH per mg of protein.

**Figure 4.** ROS levels in primary rat astroglial cell cultures at 14 DIV: exposed to glutamate 500 μM for 24 h. Bar 1: control. Bar 2: cell culture exposed 500 μM. Bar 3: cell culture exposed 500 μM plus SN1 0.5 mg/mL. Bar 4: cell culture exposed 500 μM plus SN1 1 mg/mL. Bar 5: cell culture exposed 500 μM plus SN2 0.5 mg/mL. Bar 6: cell culture exposed 500 μM plus SN2 1 mg/mL. Four replicates were carried out for each sample. (\* *p* < 0.05 and \*\* *p* < 0.001). Axis: x label: concentrations, y label: percentage of fluorescence intensity per mg protein *versus* control.
