**3. Discussion**

The present work demonstrated that pretreatments with methanol lichen extracts from cetrarioid clade provide neuroprotection against hydrogen peroxide in SH-SY5Y cells as evidenced in ROS reduction, improvement in oxidative stress biomarkers and antioxidant enzyme activity and mitochondrial protection.

The brain is a metabolically active organ that yields high ROS levels compared to other organs. Moreover, neurons are the most sensitive cell types to free radicals [11]. Overproduction of ROS can lead to oxidative macromolecules injury and consequently, to cell death. Activation of necrotic and apoptotic pathways by ROS induces cell death. Among the mechanisms responsible for ROS-causing apoptosis are receptor activation, caspase activation and mitochondrial dysfunction [12]. Oxidative stress (ROS/antioxidant imbalance) has been implicated in the initiation and progression of age-related neurodegenerative diseases [13]. Therefore, the prevention of oxidative stress is one of the most promising strategies for all those diseases that involve an alteration of redox homeostasis. H2O2 acts as an inducer of oxidative stress damage, increasing ROS levels and leading to cell death. The current study found that methanol lichen extracts from cetrarioid clade significantly attenuated hydrogen peroxide-induced ROS production in the human neuroblastoma SH-SY5Y cell line and consequently prevented cell death. Hydrogen peroxide can cross cell membranes and cause oxygen-derived free radicals. Hence, hydrogen peroxide can be converted into hydroxyl radicals in the presence of ferrous ions (Fenton reaction) [14]. Lichens contain phenolic compounds in their composition which can act as antioxidants through mechanisms such as radical scavenging activity and metal chelating activity [15–17].

Furthermore, these methanol lichen extracts mitigated changes in biomarkers of oxidative stress (lipid peroxidation reduction and GSH increase). The brain presents the highest rate of lipid metabolism in the body. In Alzheimer's disease and in Parkinson's diseases, there is an overproduction of ROS that induces the oxidation of lipid membrane constituents, leading to lipid hydroperoxides. This process, named lipid peroxidation, constitutes a hallmark for most neurodegenerative disorders. In fact, polyunsaturated fatty acids (PUFAs) are the main target of ROS attack, due to the high number of double bonds in their structure [18,19]. This early event in the brain causes cytotoxic and genotoxic effects. TBARS is a common biomarker of polyunsaturated fatty acids peroxidation. High amounts of lipid peroxidation products have been identified in post-mortem brains of people affected with neurodegenerative diseases. These lipid peroxidation products cause tissue injury and failures of antioxidant systems [20]. The lichen extracts of *V. pinastri* and *D. arctica* markedly reduced lipid peroxidation in neuroblastoma cells. Glutathione (GSH) is the major endogenous antioxidant defense. This primary antioxidant scavenges free radicals through its thiol group of its cysteine residue, and it functions as a co-substrate of the antioxidant enzymes selenium-glutathione peroxidase (GPx) and glutathione S-transferase (GST) [21]. GPx reduces lipid peroxides to alcohols and aldehydes. It has been reported that a reduction of GSH to its oxidized form provokes a decrease in intracellular GSH [22]. Restoring the levels of GSH is strongly related to the ROS and lipid peroxidation reduction [18]. Previous studies reported that fumarprotocetraric acid (depsidone), evernic acid (depside) and usnic acid (dibenzofuran-like) inhibited ROS generation, lipid peroxidation and glutathione depletion in neurons and the astrocytes cell model using hydrogen peroxide as an oxidative stress inductor [23,24]. In addition, the Parmeliaceae lichens *Cetraria islandica* and *Vulpicida canadensis* also showed protective effects against H2O2-induced injury in the human astrocytoma cell line U373-MG, as evidenced by reduced ROS production, increased GSH levels and the inhibition of lipid peroxidation [9].

Moreover, lichen extracts increase SOD and CAT activity. The enzyme SOD catalyzes the dismutation of superoxide anion to hydrogen peroxide which is then converted into oxygen and water by the action of the antioxidant enzymes catalase and glutathione peroxidase. The enzymatic activity of SOD and CAT showed to be significantly reduced in postmortem brain tissue at an advanced age [22]. Therefore, the results of this study showed that of the four methanol lichen extracts tested, *N. stracheyi* exerted its neuroprotective activity via ROS inhibition and increased CAT activity while *V. pinastri*, *D. arctica,* and *T. americana* prevented ROS overproduction and maintained enzymes activity. Upregulation of enzymes activity could be associated with an increase in its expression. Previously, it has been demonstrated that the depside fumarprotocetraric acid, isolated from *Cetraria islandica,* upregulated the antioxidant enzymes catalase, superoxide dismutase-1, and hemoxigenase1 expression which was related to Nrf2 signaling pathway activation [23]. Other lichens such as *Parmotrema perlatum* and *Hypotrachyna formosana* have also evidenced to reduce intracellular ROS generation, inhibit the peroxidation of lipids, and increase GSH levels and SOD activity [25].

Mitochondria are a major cellular organelle that play a key role in aging and degenerative diseases and are a target for oxidative damage. Mitochondria are the main source of ROS, particularly of superoxide radicals, through the complexes I and III of the respiratory chain [26,27]. An overproduction of mitochondria ROS may alter membrane permeability and calcium homeostasis as well as induce DNA mutations and injure the mitochondrial respiratory chain [28]. In our study, methanol lichen extracts of *V. pinastri* and *D. arctica* prevented mitochondrial changes by regulating calcium homeostasis and increasing mitochondrial membrane potential, suggesting a protective activity against H2O2. These extracts which target mitochondria are of great interest because they can pass across the mitochondrial phospholipid bilayer and reduce ROS damage at the heart of the source [29]. Other studies demonstrated that the depsidone fumarprotocetraric isolated from *Cetraria islandica* prevented mitochondrial membrane potential dissipation and mitochondrial calcium increase [9].

The analytical study by HPLC-UV revealed that the major compounds presented in *V. pinastri* were usnic acid, pinastric acid and vulpinic acid, and in *D. arctica* were gyrophoric acid, lecanoric acid and usnic acid, while in *T. americana* it was alectoronic acid. Lichen compounds are biosynthesized through three pathways: via the acetylpolymanolate pathway which produces depsides, depsidones and dibenzofurans, the shikimic pathway which produces pulvinic acids and the mevalonic acid pathway which is involved in terpenes formation. Therefore, gyrophoric acid and lecanoric acid are depsides, usnic acid is a dibenzofuran, alectoronic acid is a depsidone and vulpinic acid and pinastric acid are pulvinic acids [30,31]. All these lichen compounds have shown a great diversity of activities including anti-cancer (i.e., gyrophoric acid, vulpinic acid), antimicrobial (i.e., gyrophoric acid, usnic acid, vulpinic acid) photoprotective (i.e., gyrophoric acid) and neuroprotective (i.e., usnic acid) activities [32–37].

Based on the chemical structure of lichen compounds and its potential antioxidant activity, the depsides gyrophoric acid and lecanoric acid have carboxyl and hydroxyl groups that interact with several enzymatic active sites. Moreover, the aromatic rings of gyrophoric acid and lecanoric acid are responsible for their free radical scavenging properties [38,39]. Among the lichens investigated in this study, *D. artica* was the most active specie. Previous works have shown that *D. artica* has potent antioxidant properties (ORAC value 8.2 μmol TE/mg dry extract, DPPH value IC50 346.3 μg/mL and FRAP value 29.6 μmol of Fe2+ eq/g sample) which are attributed to the anti-free radical properties of gyrophoric acid and lecanoric acid [10].

On the other hand, the antioxidant properties of *V. pinastri* are mainly due to the presence of vulpinic acid and pinastric acid. These pulvinic acids have a butanolide ring with an -OH group at the 4-position and a carboxylic acid function at the double bond. This double bond is involved in radical stabilization, and is a good descriptor of antioxidant properties [17].The antioxidant activity of pulvinic acids has been demonstrated using quantitative structure–activity relationship (QSAR) techniques combined with a multivariate analysis [40,41].

Regarding lichen compounds with a depsidone structure, previous studies revealed that they are potent hydroxyl and superoxide anion radical scavengers in polar environments but not good peroxyl radical scavengers [15]. Moreover, a better hydrogen-donating potency in those depsidones with no butyrolactone ring has been reported; this is the case of alectoronic acid, which has been identified in *T. americana* [42]. Finally, the compound usnic acid, presented in *D. arctica* and *V. pinastri*, has shown reducing potential in DPPH, ABTS and DMPD radical cation assays, and superoxide radical and peroxyl scavenging abilities [16,37]. In addition to this, the presence of a phenolic ring with functional groups of −CO, −COH and −COOH showed metal chelating ability, including Fe2+ ion [16].
