*2.1. Lichen Extracts from Cetrarioid Clade Promoted Neuronal Survival after H2O2-Induced Oxidative Stress*

Initially, we evaluated the effect of the methanol extracts of the lichens *Dactylina arctica*, *Nephromopsis stracheyi, Tuckermannopsis americana* and *Vulpicida pinastri* on the human neuroblastoma SH-SY5Y cell viability using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. As shown in Figure 1, *T. americana* did not cause cytotoxicity at any assayed concentrations. On the other hand, *N. stracheyi* significantly reduced cell viability at 50 μg/mL (38% of cell viability) whereas *D. arctica* and *V. pinastri* affected cell viability at 25 μg/mL (62.7% and 65.9% of cell viability, respectively) and at 50 μg/mL (43.9% and 52.8% of cell viability, respectively).

Next, we investigated the potential protective effect of non-toxic concentrations of lichen extracts against hydrogen peroxide-induced oxidative stress. Figure 2A demonstrates that cell viability of the 250 μM for 1 h H2O2-treated SH-SY5Y cells significantly decreased by 57.5% compared to control cells (100%). However, pretreatments with methanol lichen extracts of cetrarioid clade promoted neuronal survival compared to hydrogen peroxidetreated cells. With 24 h pretreatment, the percentage of cell viability was increased over 68.9% and 76.8% for *D. arctica* at 5 and 10 μg/mL, respectively, over 65.4% and 63.2% for *N. stracheyi* at 10 and 25 μg/mL, respectively, 58.2% for *T. americana* at 50 μg/mL, and 78.9% for *V. pinastri* at 5 μg/mL. Therefore, we chose the most protective concentrations of each lichen extract to delve into the protective mechanism of these extracts and identify which of them is the most active. Hence, the maximum cell viability protection was 5 μg/mL for *V. pinastri*, 10 μg/mL for *D. arctica* and *N. stracheyi* and 50 μg/mL for *T. americana*.

Figure 2B showed the effect of the most protective lichen extracts on cell morphology. Hydrogen peroxide (250 μM for 1 h) caused morphological changes toward a cellular SH-SY5Y rounding. By contrast, lichen extracts improved morphological changes of neuroblastoma cells as shown in the presence of cellular projections.

**Figure 1.** Effect of methanol lichen extracts of cetrarioid clade on cell viability. SH-SY5Y cells were treated with different concentrations of extracts from 5 to 50 μg/mL for 24 h. Cell viability was determined using MTT assay. Results are expressed as mean ± standard deviation (SD) (triplicate experiments). \* *p* < 0.05 versus control.
