**4. Discussion**

In this study, we assessed the antioxidant effect of SN1 and SN2 extracts of *Solanum nigrum* L. leaves, both in an in vitro cellular free system and in vitro cellular models.

Oxidative stress is the causative agen<sup>t</sup> in a number of human diseases, such as atherosclerosis, ischemic reperfusion injury, inflammation, carcinogenesis, aging, and neurodegenerative diseases. Although there are many determinants in the development of these diseases, considerable experimental evidence links the production of ROS to biological damage that can potentially provide a mechanistic basis for their initiation and/or progression [36–39]. Moreover, because the ROS production is the fatal consequence of aerobic life, it is also an important component of the signaling network of plants [39], where polyphenols are the secondary metabolites produced as a defense mechanism against stress factors.

In this study, we exploited the sources, composition, and mechanisms of action of two polar *Solanum nigrum* L. leaf extracts natural products, and food, which represent a new frontier for therapy and a valuable tool to reduce the costs of health care systems.

In recent years, there has been grea<sup>t</sup> interest in the health effects of various natural products and in the in vivo protective function of natural antioxidants contained in dietary food against oxidative damage caused by ROS [40–45].

The free radical-scavenging activity is measured by the ability to bleach the stable DPPH radical. This assay provided information on the reactivity of test compounds with a stable free radical. SN1 and SN2 extracts were able to quench the DPPH-radical in a dose-dependent manner and showed comparable capacity. In fact, the two polar extracts content of different level phenolic components, but phenolic and flavones are not significantly different between SN1 and SN2. Only gentisic acid is more abundant in SN2. In addition, their effect appeared similar to Trolox. Then, this set of experiments demonstrate that SN1 and SN 2 possess comparable antioxidant properties.

Furthermore, we assessed the effect of the extracts in an in vitro cellular experimental model of excitotoxicity. In our previous research studies, using an experimental model of excitotoxicity, we demonstrated that glutamate exposure in primary cultures of astrocytes might be part of the biochemical response to oxidative stress induced by a prolonged exposure of astrocyte cultures to the neurotransmitter [46].

The antioxidant effect of the extracts SN1 and SN2 was also assessed in the cellular system using primary rat astroglial cell cultures exposed to the astroglial cell cultures in the presence of 500 μM glutamate for 24 hours. We used glutamate as a stressor because its high levels induce alterations in glutamate transport, mitochondria impairment, decrease ATP levels, GSH depletion, ROS production, macromolecular synthesis [35], and subsequent neuronal cell death [47,48].

Figure 2 shows the quenching of DPPH of SN1 and SN2 extracts at different concentrations, compared to Trolox, which shows a stronger activity at a lower concentration (0.025–0.5 and 0.1 mg/mL). In fact, we found that the extracts SN1 and SN2 were able to counteract the effect of glutamate, restoring, in a dose-dependent manner, the GSH and ROS levels similar to the control values.

The statistical analysis method in this study indicated high significance (\* *p* < 0.05 and \*\* *p* < 0.001) when compared with the control group, as reported in Figures 3 and 4, where SN1 and SN2 extracts are compared with the cells exposed to glutamate 500 μM only.

The protective effect against glutamate toxicity of the extracts SN1 and SN2 appeared stronger than that of the synthetic antioxidant compounds used in our previous research studies [46].

Thus, these findings show that the extracts SN1 and SN2 possess antioxidant properties. Furthermore, it is possible to assume that the extracts of SN1 and SN2 of *Solanum nigrum* are able to counteract glutamate uptake-induced impairment of cystine/glutamate antiporter, which leads to depletion of the GSH content and biochemical alterations. This results in the delayed toxic effect for primary astroglial cell cultures [35]. Moreover, in a previous study, we reported that a pre-incubation with GYKI 52466, the selective inhibitor of AMPA/KA receptors, diminished glutamate effects, which indicates the involvement of receptor-linked events in GSH decrease and ROS increase levels [49].

Spectrophotometric and chromatographic analytical methods applied for estimation of total phenolic content and for determination of phenolic acids and flavones compounds in the examined extracts showed that these constituents are present in a valuable amount. Our results strongly sugges<sup>t</sup> that phenolic compounds are important components of SN, and some of their pharmacological effects could be attributed to the presence of these compounds.
