Metabolomic Profiling, Antioxidant and Enzyme Inhibition Properties and Molecular Docking Analysis of Antarctic Lichens

The lichen species Lecania brialmontii, Pseudephebe pubescens, and Sphaerophorus globosus are part of the prominent lichenoflora of the Antarctic territory. In this work, we report the metabolomic identification of ethanolic extracts of these species, their antioxidant and cholinesterase enzyme inhibitory activity, and conduct a molecular docking analysis with typical compounds. Eighteen compounds were identified by UHPLC-ESI-QTOF-MS in L. brialmontii, 18 compounds in P. pubescens, and 14 compounds in S. globosus. The content of phenolic compounds was variable among the species, ranging from 0.279 to 2.821 mg AG/g, and all three species showed high inhibition potential on the cholinesterase enzymes. Molecular docking showed important interactions between AChE and BChE with the selected compounds. This study evidences the chemical fingerprint of three species of the order Lecanorales that support the continuation of the study of other biological activities and their potential for medical research.


Introduction
Lichens are a symbiotic association formed by the interaction between a photosynthetic organism (algae or cyanobacteria), an ascomycete or basidiomycete fungus, and the association of a microbiome [1,2], and, due to their ecological strategy of symbiosis, they can be found in a variety of environments, from deserts to rainforests and moors, and even in aquatic ecosystems [3]. These organisms can develop on different substrates, including bark, rock, soil, and leaves, but much of the water and nutrients are taken directly from the atmosphere [4]. Their general physiology, resulting from the characteristics of symbionts, has allowed them to be recognized as indicators of environmental quality, due to their high sensitivity to changes in atmospheric components, which affect their abundance, distribution, and vitality [5].
The Antarctic continent has an area of approximately 13,500,000 km 2 , mostly covered by permanent ice caps. Plant and fungal organisms develop to a limited extent in coastal sectors free of ice and snow during the summer period, and in rocky outcrops in the interior of the territory. Lichens are the group with the greatest specific diversity and adaptation to Antarctic conditions, due to their psychrophilic capacity that allows them to carry out biochemical and metabolic processes [6,7]. As for lichen species of the order Lecanorales, interior of the territory. Lichens are the group with the greatest specific diversity adaptation to Antarctic conditions, due to their psychrophilic capacity that allows to carry out biochemical and metabolic processes [6,7]. As for lichen species of the Lecanorales, distributed in the archipelago of the South Shetland Islands (Mar Antarctica), we find Lecania brialmontii (Vain.) Zahlbr (family Ramalinaceae), with d fruticose thallus, 6 to 13 mm high, cylindrical, lateral apothecia, with continuo crenulate margin, brownish-dark disc, saxicolous. We also find Pseudephebe pubescen M. Choisy (family Parmeliaceae), with fruiting thallus, cylindrical, black, dichotomous branches, flat at the ends, saxicolous and Sphaerophorus globosus (H Vain. (family Sphaerophoraceae), with fruticulose thallus, forming colonies, symp ramifications, on the ground between mosses and lichens. There are limited studi these species, despite their representing a source of knowledge at the taxon ecological and biological levels of the lichen community present on the white con ( Figure 1) [6,[8][9][10].  Recent research in the area of medicinal chemistry has focused its efforts on finding natural compounds from species of multiple lineages and taxa with high biological potential at the level of extracts or bioactive compounds. In lichens, phenolic-type compounds, such as depsides, depsidones, depsones, lactones, dibenzofurans, anthraquinones, pulvinic acid derivatives and xanthones, have been especially identified [11][12][13], evidencing activities of antioxidant, anti-inflammatory, anticancer, antimutagenic, and antifungal types, among others [14][15][16]. The potential of these compounds to counteract oxidative stress was reported in [17][18][19], highlighting the action of atranorin, usnic acid, lecanoric acid, diffractaic acid, lobaric acid, stictic acid, furmarprotocetraric acid, salazinic acid, physodic acid, evernic acid, gyrophoric acid, 3-hydroxyphysodic acid, physodalic acid, vesuvianic acid, sekikaic acid, isidiophorin, rhizonyl alcohol, atranol, chloroatranol, orsellinic acid and other orsellinates, mostly present in lichen species of the families Cladoniaceae, Parmeliaceae, Lobariaceae, Lecanoraceae, Ramalinaceae, Umbilicariacea and others [20]. The antioxidant action is variable, according to the level of solubility of the extract used for its evaluation and isolation of compounds, as well as the presence of components, such as proteins and carbohydrates, and is evaluated through correlation with assays, such as DPPH, ORAC, and the determination of total phenol content [21,22]. Furthermore, the evaluation of inhibitory activity on cholinesterase enzymes (AChE and BChE), as a therapeutic target for neurodegenerative diseases, was reported in species of the genera Allocetraria, Asahinea, Cetraria, Dactylina, Nephromopsis, Tuckermannopsis, Tuckneraria and Vulpicida [23].
In recent years, for the characterization of secondary metabolites in lichen extracts, the ultra-high-resolution chromatography (UHPLC) technique, coupled to a mass spectrometer, has been used to obtain diagnostic fragments for each type of compound, forming a robust and accurate tool for the chemical study of lichen extracts [24][25][26][27][28]. In this work, we report the metabolomic identification of the ethanolic extracts of L. brialmontii, P. pubescens, and S. globosus, their antioxidant and cholinesterase enzyme inhibitory activity, and conduct a molecular docking analysis with typical compounds in the study species. Virtual structurebased screening (VS) is a widely used process, that uses the properties and knowledge of the three-dimensional structure of the protein of interest with the aim of designing new organic molecules to be used as potential drugs [29]. This process evaluates, through predictions, the possible unions and affinities of the organic molecules within the binding site of the protein of interest, which facilitates an understanding of the important properties related to the binding process. Empirical scoring functions are widely used for posing and affinity prediction [29,30]. Although pose prediction is performed with satisfactory accuracy, correct binding affinity accuracy remains a challenging and crucial task for the success of structure-based VS experiments. However, these scores allow evaluation of the possible behavior of the organic molecule at the protein binding site. We know that for the study of molecular coupling there are various software that can be used, that can have a higher performance in the identification and qualification of molecular coupling [31]. Therefore, in this study, it was decided to perform the molecular docking analyses at UCSF Chimera using Autodock Vina tools. The fingerprinting of the ethanolic extract of L. brialmontii was obtained by means of high-resolution mass spectrometric analysis (UHPLC-MS). The negative mode was used for the tentative identification of the eighteen peaks ( Figure 2). The metabolites identified in this species were carbohydrates, acids, anthraquinones, aromatics, lipids and depsides (Table 1).

Results and Discussion
properties and knowledge of the three-dimensional structure of the protein of interest with the aim of designing new organic molecules to be used as potential drugs [29]. This process evaluates, through predictions, the possible unions and affinities of the organic molecules within the binding site of the protein of interest, which facilitates an understanding of the important properties related to the binding process. Empirical scoring functions are widely used for posing and affinity prediction [29,30]. Although pose prediction is performed with satisfactory accuracy, correct binding affinity accuracy remains a challenging and crucial task for the success of structure-based VS experiments. However, these scores allow evaluation of the possible behavior of the organic molecule at the protein binding site. We know that for the study of molecular coupling there are various software that can be used, that can have a higher performance in the identification and qualification of molecular coupling [31]. Therefore, in this study, it was decided to perform the molecular docking analyses at UCSF Chimera using Autodock Vina tools.

UHPLC Chromatographic Analysis of Lecania brialmontii
The fingerprinting of the ethanolic extract of L. brialmontii was obtained by means of high-resolution mass spectrometric analysis (UHPLC-MS). The negative mode was used for the tentative identification of the eighteen peaks ( Figure 2). The metabolites identified in this species were carbohydrates, acids, anthraquinones, aromatics, lipids and depsides (Table 1).
The genus Lecania has been a focus of study to objectively define its species through molecular phylogeny studies using nucleotide sequences from the mt-SSU rRNA, the ITS region of the nu-rDNA, and the RNA polymerase II second largest subunit [34]. However, the characterization of the chemical fingerprint of the species could contribute to elucidate their similarities and differences and new groupings. On the other hand, L. brialmontii, together with species of the genera Usnea, Polycauliona and Cladonia, have been reported as a source of leavenings that produce extracellular hydrolytic enzymes and are of interest in biotechnological applications of catalytic origin [35].

UHPLC Chromatographic Analysis of Pseudephebe pubescens
The fingerprinting of the ethanolic extract of P. pubescens was obtained by means of high-resolution mass spectrometric analysis (UHPLC-MS). The negative mode was used for the tentative identification of the eighteen peaks ( Figure 2). The metabolites identified in this species were carbohydrates, acids, lipids, depsides, aromatics and dibenzofurans ( Table 2).
Carbohydrates Peak 1 was identified as mannitol (C 6 H 13 O 6 ). Peak 4, with a molecular anion at m/z 245.0484, was identified as visnagin (C 13 H 9 O 5 ); while peak 5 was identified, with an [M−H] − ion at m/z 245.0489, as the related khellinol (C 13 H 9 O 5 ).

Fatty Acids
Peak 2 was identified as citric acid (C 6 H 7 O 7 ).

Lipids
Peak 3 was identified as azelaic acid (C 9 H 15 O 4 ); while peak 6 was identified as 9octadecenedioic acid (C 18 H 31 O 4 ) and peak 7 was tentatively identified as pinellic acid (C 18  , with diagnostic peaks at m/z 295.2291, 231.0647, and 328.0570. This compound was reported in previous studies and is recognized for multiple biological activities in in vitro and in vivo assays [36].
The genus Pseudephebe has been of great interest for studies of species delimitation and biogeographic processes, based on molecular tools and microevolutionary analyses that facilitate the defining of the origin of its distribution [37]. Therefore, these species represent a model for the study of biological, ecological, chemical, and systematic variables.

UHPLC Chromatographic Analysis of Sphaerophorus globosus
The fingerprinting of the ethanolic extract of S. globosus was obtained by means of high-resolution mass spectrometric analysis (UHPLC-MS). The negative mode was used for the tentative identification of the fourteen peaks ( Figure 2). The metabolites identified in this species were carbohydrates, aromatics, acids, depsides and dibenzofurans (Table 3).   [32][33][34]36]. The compound sphaerophorin is reported to have a protective effect against human melanoma cells, inhibiting their growth and promoting apoptotic cell death [38].
In general, previous studies of the chemical composition of S. globosus extracts have evidenced chemopreventive potential in cancer, such as blocking estrogen formation by inhibition of the aromatase enzyme [39]. Likewise, the intervention of this lichen in primary colonization processes [40], exhibits a potential for further study, to correlate the presence of compounds with ecological advantages.

Total Phenolic Contents and Antioxidant Activity
The range of polyphenolic content present in ethanol extract in the three lichen species ranged from 0.279 to 2.821 mg AG/g and showed moderate antioxidant activity ( Table 4). The species P. pubescens showed a higher presence of phenolic compounds. On the other hand, S. globosus presented a higher oxygen radical absorption capacity, compared to the higher reducing power of P. pubescens.
The total phenolic content was relatively low. However, these results coincided with previous reports where species such as P. pubescens showed low content of phenolic compounds, but high concentrations of polysaccharides and proteins [41]. The antioxidant activity of the ethanolic extracts was high, and in species such as P. pubescens and S. globosus, high values of superoxide anion trapping were previously reported with methanolic and acetone extracts [42]. These results are in addition to the significant antioxidant capacity reported in extracts and compounds of lichen species of the genera Toninia, Usnea, Parmelia, Flavoparmelia, Evernia, Hypogymnia, Cladonia, Vulpicida, Pseudevernia and Cetraria [19,[43][44][45].

Enzymatic Inhibitory Activity
The values obtained with the ethanolic extracts of the three lichen species (Table 5), showed high inhibition potential on the cholinesterase enzymes. For AChE, the inhibition ranged from 3949 to 10,422 µg/mL, while for BChE it ranged from 4476 to 8828 µg/mL. These results are comparable with the work reported with the ethanolic extract of the Antarctic species Himantormia lugubris in [28], which showed an inhibitory activity on AChE of 12.38 ± 0.09 µg/mL and on BChE of 31.54 ± 0.20 µg/mL, while the ethanolic extracts of L. brialmontii, P. pubescens and S. globosus showed a higher potential for inhibition of cholinesterase enzymes. These data reported better inhibition efficacy against Cladonia uncinalis extract, which weakly inhibited AChE, and, for BChE, the extracts of Parmelia sulcata and C. uncinalis showed inhibition values of 42.9 ± 0.1 µg/mL and 85.9 ± 0.2 µg/mL, respectively [46]. On the other hand, the inhibiting activity in the endogenous fungus Diaporthe mehothocarpus from the lichen Cladonia symphycarpia [47] was much lower compared to the results obtained in the three study species.

Docking Studies
The compounds present and typical in the extracts of Lecania brialmontii, Pseudephebe pubescens and Sphaerophorus globosus, as well as the known inhibitor of acetylcholinesterase and butyrylcholinesterase, were used for molecular docking assays in order to analyze the protein molecular interactions with the main amino acid residues involved in the inhibition of these structures. The best coupling binding affinities for each of the ligands were expressed in kcal/mol and compared with the coupling energy of the inhibitor galantamine (Table 6).  Table 6 shows the binding affinities of the compounds barbatic acid, lecanoric acid, brialmontin 2, tetrahydroxytricosanoic acid, sphaerophorin and sekikaic acid obtained from the extracts of Lecania brialmontii, Pseudephebe pubescens and Sphaerophorus globosus. Most of these compounds presented good energy descriptors on the enzyme acetylcholinesterase. Barbatic acid and sphaerophorin were arranged in the same way at the catalytic site of acetylcholinesterase ( Figure 3A,E), which could probably explain the similarity in their binding affinities (−10.30 kcal/mol and −9.50 kcal/mol, respectively). Likewise, both compounds presented the same hydrogen bonds between the carbonyl group of the carboxylate in their structures and the residues Ser200, Gly118 and Gly119 ( Figure 4A,E). Similarly, it was observed that the two compounds interacted with the main residues in the catalytic site, which were Ser200 and His400; thus, allowing greater stability in the catalytic site of acetylcholinesterase. However, barbatic acid presented a binding affinity very similar to that of the reference inhibitor galantamine. In addition to the interactions of hydrogen bonds that the barbatic acid presented, it showed π-π and π-sigma type interactions with the Tyr334 and Phe331 residues, respectively ( Figure 4A). hydroxyl group of the aromatic ring and the His400 residue ( Figures 3B and 4B), which is one of the important residues in the catalytic site of acetylcholinesterase, giving it high affinity and, therefore, high binding affinity.  Tetrahydroxytricosanoic acid had the lowest binding affinity (Table 6); this was due to the number of rotatable bonds that its molecular structure presented, causing its molecular geometry to be less stable in the catalytic site of acetylcholinesterase. It was observed that the tetrahydroxytricosanoic acid presented 2 interactions of hydrogen bonds between the oxygen atom of the carboxylate and the Ser122 residue, and the alpha carbon hydroxyl group with the Asp72 residue ( Figures 3D and 4D). However, the interactions that the aliphatic chain of tetrahydroxytricosanoic acid presented were very weak, which meant that it did not have good disposition and stability in the binding site with acetylcholinesterase.
Brialmontin 2 and sekikaic acid were arranged in a very similar way in the catalytic site of acetylcholinesterase ( Figure 3C,F), and very similar binding energies (−9.80 kcal/mol and −9.30 kcal/mol) and very similar interactions with some amino acid residues was observed. However, these two compounds presented some differences. The two compounds presented hydrogen bond type interactions with the Tyr121 residue. However, brialmontin 2 did so with the oxygen atom of the ester group of its structure, while sekikaic acid performed the interaction with the oxygen atom of the group ester carbonyl ( Figure 4C,F). These two compounds presented π-π T-shaped interactions with the Phe330 amino acid, but with the difference that in brialmontin 2 it was present between the methoxyl group of the aromatic ring and the aromatic ring of the Phe330 residue, while with sekikaic acid it was carried out between the ring aromatic part of its structure and the aromatic ring of the Phe330 residue.
Lecanoric acid was the second compound that presented a higher binding affinity for acetylcholinesterase because it presented hydrogen bond type interactions between the hydroxyl group of the aromatic ring and the His400 residue ( Figures 3B and 4B), which is one of the important residues in the catalytic site of acetylcholinesterase, giving it high affinity and, therefore, high binding affinity.

Butyrylcholinesterase (hBChE) Docking Results
The molecular isolation results between the selected compounds of the extract of Lecania brialmontii, Pseudephebe pubesecens and Sphaerophorus globosus and butyrylcholinesterase (hBChE) showed lower binding affinities, compared to the results obtained with acetylcholinesterase (Table 6). It was observed that most of the compounds exhibited slightly higher binding affinities compared to the reference inhibitor galantamine. However, tetrahydroxytricosanoic acid stood out as the compound with the lowest binding affinity compared to the other compounds (Table 6) and this behavior was observed in the same way in the results obtained with acetylcholinesterase (TcAChE). As mentioned above, this exchange was highly influenced by the stability of the structure, since having a high number of rotatable bonds prevented it from having a better

Butyrylcholinesterase (hBChE) Docking Results
The molecular isolation results between the selected compounds of the extract of Lecania brialmontii, Pseudephebe pubesecens and Sphaerophorus globosus and butyrylcholinesterase (hBChE) showed lower binding affinities, compared to the results obtained with acetylcholinesterase (Table 6). It was observed that most of the compounds exhibited slightly higher binding affinities compared to the reference inhibitor galantamine. However, tetrahy-droxytricosanoic acid stood out as the compound with the lowest binding affinity compared to the other compounds (Table 6) and this behavior was observed in the same way in the results obtained with acetylcholinesterase (TcAChE). As mentioned above, this exchange was highly influenced by the stability of the structure, since having a high number of rotatable bonds prevented it from having a better conformation in the catalytic site of butyrylcholinesterase (hBChE) ( Figure 5D). However, it was observed that tetrahydroxytricosanoic acid showed hydrogen bonds with residues Ser198 and Gly117 ( Figure 6D). Although these residues were involved in the inhibition of butyrylcholinesterase (hBChE), the instability of tetrahydroxytricosanoic acid caused presentation of a lower binding affinity compared to the other compounds. Lecanoric acid showed better binding affinity results with butyrylcholinesterase (hBChE), due to its interactions with the main residues of the catalytic site responsible for inhibition of butyrylcholinesterase (hBChE). Figure 6B shows that lecanoric acid exhibited 4 hydrogen bonds, one of which was between the oxygen atom of the carboxylate and the hydrogen of Ser198. The next hydrogen bond interaction was between the carbonyl group of the carboxylate and the Tyr128 residue, and another hydrogen bond interaction was between the group phenolic ring hydroxide and the Pro285 residue. The last hydrogen bonding interaction was between the ester cabonyl group with the Tyr332 residue. These interactions favored the blocking of lecanoric acid within the catalytic site ( Figure 5B), which generated greater stability and, therefore, a better binding affinity with butyrylcholinesterase (hBChE). methyl of the group methoxyl with residue Trp82 (Figures 5C and 6C). Figures 5F and 6F show the interactions that sekikaic acid presented with butyrylcholinesterase (hBChE). Hydrogen bond type interactions were present between the hydroxyl of the aromatic ring with the residue Thr120. Signals of π-sigma type were observed between the methyl groups of the propyl attached to the aromatic rings and the residues Trp231 and Tyr332 and π-π interactions were present between residues Gly116 and Trp82 with the aromatic rings ( Figures 5F and 6F).  Lecanoric acid showed better binding affinity results with butyrylcholinesterase (hBChE), due to its interactions with the main residues of the catalytic site responsible for inhibition of butyrylcholinesterase (hBChE). Figure 6B shows that lecanoric acid exhibited 4 hydrogen bonds, one of which was between the oxygen atom of the carboxylate and the hydrogen of Ser198. The next hydrogen bond interaction was between the carbonyl group of the carboxylate and the Tyr128 residue, and another hydrogen bond interaction was between the group phenolic ring hydroxide and the Pro285 residue. The last hydrogen bonding interaction was between the ester cabonyl group with the Tyr332 residue. These interactions favored the blocking of lecanoric acid within the catalytic site ( Figure 5B), which generated greater stability and, therefore, a better binding affinity with butyrylcholinesterase (hBChE).
Barbatic acid had a lower binding affinity for butyrylcholinesterase (hBChE) than for acetylcholinesterase (TcAChE). This was, mainly because the carboxylate group had a negative interaction with the Glu197 residue ( Figure 6A), which caused its stability to decrease at the site catalytic of butyrylcholinesterase (hBChE), so it presented lower binding affinity. However, it was observed that it showed hydrogen bond type interactions with one of the residues responsible for the inhibition of butyrylcholinesterase (hBChE), which was the Ser198 residue ( Figures 5A and 6A).
Sphaerophorin and galantamine were shown to have very similar binding affinities (−8.70 kcal/mol and −8.80 kcal/mol, respectively), since sphaerophorin interacted well with the catalytic site of butyrylcholinesterase (hBChE) (Figures 5E and 6E). As in the other compounds, the carboxylate group presented favorable interaction of the hydrogen bond type with the residues Ser198 and Tyr128 ( Figure 6E). The phenolic ring hydroxyl group exhibited two hydrogen bonding interactions with residues Tyr332 and Asp70. Interactions of the π-sigma type were observed between the methoxyl group of the aromatic ring and residue Phe329, and, in addition, π-π type interactions between the aromatic ring and residues His438 and Trp82 were observed ( Figure 6E). These interactions favored sphaerophorin presenting stability in the catalytic site, confirming good binding affinity ( Figure 5E and Table 6).
Brialmontin 2 presented π-sigma interactions between the methyl group of the aromatic ring and residue Tyr332, as well as carbon-hydrogen bond type interactions between the oxygen atom of the ester with residue His438, the oxygen atom and the methyl of the group methoxyl with residue Trp82 (Figures 5C and 6C). Figures 5F and 6F show the interactions that sekikaic acid presented with butyrylcholinesterase (hBChE). Hydrogen bond type interactions were present between the hydroxyl of the aromatic ring with the residue Thr120. Signals of π-sigma type were observed between the methyl groups of the propyl attached to the aromatic rings and the residues Trp231 and Tyr332 and π-π interactions were present between residues Gly116 and Trp82 with the aromatic rings ( Figures 5F and 6F).

Prediction of Pharmacokinetic Properties-ADME
The phytochemicals extracted from Lecania brialmontii, Pseudephebe pubesecens and Sphaerophorus globosus were subjected to pharmacokinetic analysis comparing them with the cholinesterase inhibitor galantamine, using the Osiris Data Warrior program (Table 7). According to Lipinski's 'rule of five', a good drug candidate for consideration in preclinical studies should have a molecular weight (MW) ≤ 500 Da, number of gyratory bonds ≤ 10, number of acceptor hydrogen bonds ≤ 10, number of hydrogen bond donors ≤ 5 and a Clog p value ≤ 5 [48,49]. As shown in Table 7, the only phytochemical that did not meet the criteria was tetrahydroxytricosanoic acid, due to its higher number of rotatable bonds and having cLogP greater than five. These results were highly related to the docking analysis, since tetrahydroxytricosanoic acid presented lower binding affinities in cholinesterases. Note: a Percentage of absorption (%ABS); b topological polar surface area (TPSA); c molecular weight (MW); d logarithm of partition coefficient between n-octanol and water (cLogP); e number of hydrogen bond donors (HBD); f number of hydrogen bond acceptors (HBA); g number of rotable bonds (n-ROTB). * Compound recognized as a standard inhibitor.
The bioavailability of the phytochemicals was evaluated by TPSA analysis. This parameter was highly related to passive molecular transport through membranes and to assessing the bioavailability of phytochemicals. From the TPSA values, the absorption percentages were calculated using Equation (1) [50]. The best absorption percentage values were presented by Brialmontin 2 (86.59%), because it presented a lower TPSA compared to the other phytochemicals. However, most of the phytochemicals presented absorption percentages greater than 60% (Table 7), indicating good bioavailability.

Toxicity Prediction
The pharmacodynamic (toxicological) properties of the phytochemicals extracted from Lecania brialmontii, Pseudephebe pubescens and Sphaerophorus globosus were evaluated using the Osiris Data Warrior computational tool. The toxicity risks that were evaluated were mutagenicity, tumorigenicity, irritation and reproductive toxicities ( Table 8). The results showed that the only phytochemicals with toxicity risks were Brialmontin 2 and tetrahydroxytricosanoic acid. Brialmontin presented high levels of tumorigenicity and irritation since, when metabolized, one of the aromatic rings that contains two methyl groups, conferred the risk of tumorigenicity (Table 9). Tetrahydroxytricosanoic acid showed low risks of reproductive toxicities and irritation, due to the high number of rotatable bonds ( Table 7) in its structure, giving it high reactivity.

Preparation of the Ethanolic Extracts
About 5 g of each lichen species was macerated with ethanol (three times, 30 mL each time) by ultrasound at room temperature. Each extract was filtered, and the solutions were concentrated under reduced pressure at 38 • C to obtain a gummy extract.

LC Parameters and MS Parameters
The separation and identification of secondary metabolites from the lichens were carried out on a UHPLC-ESI-QTOF-MS system, equipped with UHPLC Ultimate 3000 RS with Chromeleon 6.8 software (Dionex GmbH, Idstein, Germany), and a Bruker maXis ESI-QTOF-MS with the software Data Analysis 4.0 (all Bruker Daltonik GmbH, Bremen, Germany). The chromatographic equipment consisted of a quaternary pump, an autosampler, a thermostated column compartment, and a photodiode array detector. The elution was performed using a binary gradient system with eluent (A) 0.1% formic acid in the water, eluent (B) 0.1% formic acid in acetonitrile, and the gradient: isocratic 1% B (0-2 min), 1-5% B (2-3 min), isocratic 5% B (3-5 min), 5-10% B (5-8 min), 10-30% B (8-30 min), 30-95% B (319-38 min), and 1% B isocratic (39-50 min). The separation was carried out with an acclaim Thermo 5 µm C18 80 Å (150 mm × 4.6 mm) column at a flow rate of 1.0 mL/min. ESI-QTOF-MS experiments in negative ion mode were recorded and the scanning range was between 100 and 1200 m/z. Electrospray ionization (ESI) conditions included capillary temperature of 200 • C, a capillary voltage of 2.0 kV, dry gas flow of 8 L/min, and a pressure of 2 bars for the nebulizer. The experiments were performed in automatic MS/MS mode. The structural characterization of the bioactive compounds was based on HR full MS, fragmentation patterns, and similarity with literature data. For the analysis, 5 mg of each extract was dissolved in 2 mL of methanol, passed through a polytetrafluoroethylene (PTFE) filter, and 10 µL were injected into the apparatus.

Total Phenolic Content
The total phenolic content (TPC) of L. brialmontii, P. pubescens and S. globosus extracts was measured by the Folin-Ciocalteu method and the AlCl 3 method, using a Synergy HTX microplate reader (Biotek, Winoosky, VT, USA) [51]. Results were expressed as mg of gallic acid per gram of dried lichen. Each experiment was performed in triplicate and values were expressed with mean and standard deviation.
3.6. Antioxidant Activity 3.6.1. Ferric-Reducing Antioxidant Power (FRAP) Assay The FRAP assay was performed following a method previously described in [52]. The standard used was Trolox, and by interpolation on a calibration curve, the results were expressed as micromoles of Trolox equivalents per gram of dried lichen. All experiments were performed in triplicate, and the data were reported as the mean and its standard deviation.

Oxygen Radical Absorbance Capacity (ORAC) Assay
The ORAC assay was performed following a previously described method in [53]. A calibration curve was prepared with the Trolox standard in reaction with fluorescein. The results were expressed in micromoles of Trolox equivalents per gram of dry lichen. Each experiment was performed in triplicate, and the data were reported as the mean and its standard deviation.

Determination of Cholinesterase Inhibition
The determination of this activity was based on the Ellman method, reported previously in [54]. The galantamine standard was prepared to make the calibration curve. DTNB solution, AChE, and BChE enzyme solution, and the addition of substrates acetylthiocholine iodide and butyryl-thiocholine chloride, as appropriate, were used in a microplate. Each experiment was performed in triplicate and values were expressed as µg/mL denoting the IC50 for each sample.

Docking Simulations
Crystallographic enzyme structures of Torpedo Californica acetylcholinesterase (TcAChE; PDBID: code 1DX6 [55]), human butyrylcholinesterase (hBChE; PDBID: code 4BDS [56]) downloaded from the RCSB PDB protein data bank [57,58] were analyzed. Enzyme optimizations were carried out using UCSF Chimera software (v1.16, San Francisco, CA, USA), where water molecules and ligands were removed from crystallographic protein active sites. In the same way, all polar hydrogen atoms were added at pH = 7.4. The appropriate ionization states for basic and acidic amino acid residues were considered. The size of the bounding box was fixed at a cube with sides of length 20 Å. The centroid of the selected residue was chosen based on the putative catalytic site of each enzyme, considering its known catalytic amino acids: Ser200 for acetylcholinesterase (TcAChE) [59,60], Ser198 for butyrylcholinesterase (hBChE) [61,62]. The two-dimensional structures of the ligands were drawn with ChemDraw 8.0 (PerkinElmer Informatics, Waltham, MA, USA) and imported into Avogadro (https://avogadro.cc, accessed on 20 April 2022) to optimize the geometry using the force field function MMFF94 [63,64]. All compounds were saved as mol2 files for further docking studies [65][66][67]. The standard procedure for molecular coupling was performed, using the corresponding rigid crystallographic enzymatic structures and flexible ligands (Barbatic acid, Lecanoric acid, Brialmontin 2, Tetrahydroxytricosanoic acid, Sphaerophorin and Sekikaic acid), whose torsion angles were identified (for 10 independent runs per ligand). Directed coupling was performed using the UCSF Chimera program [68,69], where the catalytic pocket of the reference inhibitor galantamine for acetylcholinesterase and butyrylcholinesterase was used. Polar hydrogens and partial Gasteiger charges were added, and a grid box was created using the Autodock Vina tools at UCSF Chimera. Docking and analysis results were visualized using Discovery Studio Visualizer [70]. Upon completion of coupling, the best conformation for hydrogen bonding or π interactions, including the binding energy of the free ligand (kcal/mol), was analyzed [66,67].

Calculation of ADME Parameters
Osiris Data Warrior toolkits (v 5.5.0) were used to verify the pharmacokinetic properties of the phytochemicals extracted. Molecular descriptors that were calculated are the logarithm of the partition coefficient (cLogP), the number of hydrogen bond donors, number of hydrogen bond acceptors, molecular mass of compounds, topological polar surface area (TPSA), number of spin bonds, and violations Lipinski's rule of five. Using the TPSA value, the absorption percentage (% ABS) was calculated using the following Equation (1) [50,71]: % ABS = 109 − (0.345 × TPSA) (1)

Calculation of Risk Toxicity
To calculate the toxicological properties of the phytochemicals, the Osiris Data Warrior computational tool was used. The toxicity risks evaluated in each of the molecules were mutagenicity, tumorigenicity, irritation and reproductive effect [50,72].

Statistical Analysis
The results were expressed as the mean of the data with standard deviation (SD) using GraphPad Prism software, version 5.0. (GraphPad Software, Inc., La Jolla, CA, USA). The determination was performed in triplicate for each sample solution. Statistical significance was set at p < 0.05 and were determined by one-way ANOVA.

Conclusions
This work contributes to the chemical characterization of lichen species present in the Antarctic territory Lecania brialmontii, Pseudephebe pubescens and Sphaerophorus globosus. By UHPLC-ESI-QTOF-MS analysis, 18 compounds were identified in L. brialmontii, 18 compounds in P. pubescens and 14 compounds in S. globosus, corresponding to carbohydrates, acids, anthraquinones, aromatics, lipids, depsides and dibenzofurans. The results showed a relatively low phenolic composition in the species and moderate antioxidant activity; likewise, the cholinesterase inhibitory activity showed good values. Molecular docking analysis with typical compounds of these species showed an important interaction on AChE and BChE enzymes as possible inhibitors. In general, these species represent a promising biological resource for research in the prevention and/or neuroprotection of central nervous system diseases, such as Parkinson's and Alzheimer's disease.

Data Availability Statement:
The data presented in this study are available on request from the correspondings authors.