*2.4. Lichen Extracts from Cetrarioid Clade Protected against H2O2-Induced Mitochondrial Dysfunction*

Figure 5 shows the effect of lichen extracts on different mitochondrial parameters (mitochondrial membrane potential and calcium levels). Treatment with hydrogen peroxide (250 μM, 1 h) caused a significant decrease in the mitochondrial membrane potential (35% compared to 100% control cells) and a significant increase in mitochondrial calcium levels (1.14 relative to control) and cytosolic calcium levels (1007 nM compared to 516 nM control cells). However, pretreatments with lichen extracts prevented H2O2-induced mitochondrial changes. In particular, extracts of *D. arctica* (10 μg/mL) and *V. pinastri* (5 μg/mL) significantly increased mitochondrial membrane potential by 67% and 69%, respectively, and significantly reduced cytosolic calcium levels by 557 nM and 721 nM, respectively. Mitochondrial calcium levels were reduced in pretreated cells with selected concentrations of *D. arctica*, *V. pinastri* and *T. americana* extracts (1.08, 1.07, 1.05 relative to control, respectively).

**Figure 5.** *Cont*.

**Figure 5.** Effect of lichen extracts against H2O2 -induced mitochondrial dysfunction in SH-SY5Y. (**A**) on cytosolic calcium levels. (**B**) on mitochondrial calcium levels. (**C**) on mitochondrial membrane potential. Data are expressed as means ± SD (% of control) \* *p* < 0.001 vs. control; # *p* < 0.001 vs. H2O2).

#### *2.5. HPLC Profile of Lichen Extracts from Cetrarioid Clade*

The most promising lichen extracts were analyzed using the HPLC-UV method, whose representative chromatograms are shown in Figure 6. Secondary metabolites were identified based on their retention times and ultraviolet spectra as compared to standards and previously reported lichen extracts. Table 1 reported retention times and the absorbance maxima (nm) UV spectrum. Results showed that the main compounds in *D. artica* were gyrophoric acid (GYR) and lecanoric acid (LEC). The lichen *T. americana* contained alectoronic acid (ALE). The compounds usnic acid (USN), pinastric acid (PIN) and vulpinic acid (VUL) were the majority in *V. pinastri*.

**Table 1.** Retention times and UV absorbance maxima (nm) of main secondary metabolites of studied lichens.


**Figure 6.** Representative HPLC chromatograms (λ = 254 nm) (**A**) *Dactylina artica* (**B**) *Tuckermannopsis americana* (**C**) *Vulpicida pinastri*.
