Methodological Aspects of Green Extraction of Usnic Acid Using Natural Deep Eutectic Solvents.

Usnic acid (UA) is a compound with multiple biological activities that make it useful in various industries, e.g., pharmaceutical, cosmetic, dentistry, and agricultural sectors. Lichens are the primary source of UA, which is primarily extracted using acetone. This study aimed to investigate the solubility of UA in numerous natural deep eutectic solvents (NADESs) and use a mixture of thymol and camphor as a NADES in the optimization of the UA extraction process with the design of experiments method. For numerical optimization, the following parameters were employed in the experiment to confirm the model: a camphor-to-thymol ratio of 0.3, a liquid-to-solid ratio of 60, and a time of 30 min. The obtained experimental results aligned well with the predicted values, with the mean experimental value falling within the confidence interval, exhibiting deviations between 11.93 and 14.96. By employing this model, we were able to optimize the extraction procedure, facilitating the isolation of approximately 91% of the total UA content through a single extraction, whereas a single acetone extraction yielded only 78.4% of UA.

Antimelanoma Potential of Cladonia mitis Acetone Extracts - Comparative in Vitro Studies in Relation to Usnic Acid Content.

In this study, the cytotoxic activity of acetone extracts of Cladonia mitis was assessed with respect to the content of usnic acid, a secondary metabolite commonly present in this species. Following quantitative HPLC analysis of the extracts, usnic acid was isolated by preparative chromatography. The study of cytotoxic activity was performed using the MTT test on three melanoma cell lines - HTB140, A375 and WM793. The selectivity of action was also assessed by comparing the effect towards normal human keratinocytes HaCaT. The results showed a dose-dependent cytotoxic activity of the extracts tested and usnic acid itself, but no relationship was found between the content of usnic acid and the activity of the extracts. Furthermore, the extracts showed varied, but rather low anti-tyrosinase activity. Other in vitro and in vivo studies are necessary to demonstrate that C. mitis extracts may be useful in the adjuvant external treatment of skin melanoma.

Lichens buffer tundra microclimate more than the expanding shrub Betula nana.

BACKGROUND AND AIMS: In tundra systems, soil-borne lichens are often the dominant groundcover organisms, and act to buffer microclimate extremes within or at the surface of the soil. However, shrubs are currently expanding across tundra systems, potentially causing major shifts in the microclimate landscape. METHODS: Here, we compared soil temperature and moisture underneath the dwarf birch Betula nana and seven abundant lichen species in sub-alpine Norway. We also examined mixtures of lichens and dwarf birch - an intermediate phase of shrubification - and measured several functional traits relating to microclimate. KEY RESULTS: We found that all lichen species strongly buffered the daily temperature range, on average reducing maximum temperatures by 6.9 °C (± 0.7 s.d.) and increasing minimum temperatures by 1.0 °C (± 0.2 s.d.) during summer. The dwarf birch had a much weaker effect (maximum reduced by 2.4 ±â€…5.0 °C and minimum raised by 0.2 ± 0.9 °C). In species mixtures, the lichen effect predominated, affecting temperature extremes by more than would be expected from their abundance. Lichens also tended to reduce soil moisture, which could be explained by their ability to intercept rainfall. Our trait measurements under laboratory conditions suggest that, on average, lichens can completely absorb a 4.09 mm (± 1.81 s.d.) rainfall event, which might be an underappreciated part of lichen-vascular plant competition in areas where summer rainfall events are small. CONCLUSIONS: In the context of shrubification across tundra systems, our findings suggest that lichens will continue to have a large effect on microclimate until they are fully excluded, at which point microclimate extremes will increase greatly.

Population genomics of a reindeer lichen species from North American lichen woodlands.

PREMISE: Lichens are one of the main structural components of plant communities in the North American boreal biome. They play a pivotal role in lichen woodlands, a large ecosystem situated north of the closed-crown forest zone, and south of the forest-tundra zone. In Eastern Canada (Quebec), there is a remnant LW found 500 km south of its usual distribution range, in the Parc National des Grands-Jardins, originated mainly because of wildfires. We inferred the origin of the lichen Cladonia stellaris from this LW and assessed its genetic diversity in a postfire succession. METHODS: We genotyped 122 individuals collected across a latitudinal gradient in Quebec. Using the software Stacks, we compared four different approaches of locus selection and single-nucleotide polymorphism calling. We identified the best fitting approach to investigate population structure and estimate genetic diversity of C. stellaris. RESULTS: Populations in southern Quebec are not genetically different from those of northern LWs. The species consists of at least four phylogenetic lineages with elevated levels of genetic diversity and low co-ancestry. In Parc National des Grands-Jardins, we reported high values of genetic diversity not related with time since fire disturbance and low genetic differentiation among populations with different fire histories. CONCLUSIONS: This first population genomic study of C. stellaris is an important step forward to understand the origin and biogeographic patterns of lichen woodlands in North America. Our findings also contribute to the understanding of the effect of postfire succession on the genetic structure of the species.

Microwave-Assisted Extraction and HPLC-UV-CD Determination of (S)-usnic Acid in Cladonia foliacea.

During the years, many usnic acid (UA) conjugates have been synthesized to obtain potent endowed with biological properties. Since (S)-UA is less abundant in nature than (R)-enantiomer, it is difficult to source, thus precluding a deeper investigation. Among the lichens producing UA, Cladonia foliacea is a valuable (S)-UA source. In the present work, we report on a rapid HPLC-UV/PAD-CD protocol suitable for the analysis and the identification of the main secondary metabolites present in C. foliacea extract. Best results were achieved using XBridge Phenyl column and acetonitrile and water, which were both added with formic acid as mobile phase in gradient elution. By combining analytical, spectroscopical, and chiroptical analysis, the most abundant analyte was unambiguously identified as (S)-UA. Accordingly, a versatile microwave-assisted extractive (MAE) protocol, assisted by a design of experiment (DoE), to quantitatively recover (S)-UA was set up. The best result in terms of UA extraction yield was obtained using ethanol and heating at 80 °C under microwave irradiation for 5 min. Starting from 100 g of dried C. foliacea, 420 mg of (S)-UA were achieved. Thus, our extraction method resulted in a suitable protocol to produce (S)-UA from C. foliacea for biological and pharmaceutical investigation or commercial purposes.

Lichenicoccus roseus gen. nov., sp. nov., the first bacteriochlorophyll a-containing, psychrophilic and acidophilic Acetobacteraceae bacteriobiont of lichen Cladonia species.

Gram-negative, aerobic, chemo-organotrophic and bacteriochlorophyll a-containing bacterial strains, KEBCLARHB70RT, KAMCLST3051 and KAMCLST3152, were isolated from the thalli of Cladonia arbuscula and Cladonia stellaris lichens. Cells from the strains were coccoid and reproduced by binary division. They were motile at the early stages of growth and utilized sugars and alcohols. All strains were psychrophilic and acidophilic, capable of growth between pH 3.5 and 7.5 (optimum, pH 5.5), and at 4-30 °C (optimum, 10-15 °C). The major fatty acids were C18 : 1 ω7c and C18 : 0; the lipids were phosphatidylcholines, phosphatidylethanolamines, phosphatidic acids, phosphatidylglycerol, glycolipids, diphosphatidylglycerol and polar lipids with an unknown structure. The quinone was Q-10. The DNA G+C content was 67.8 mol%. Comparative 16S rRNA gene analysis together with other data, supported that the strains, KEBCLARHB70RT, KAMCLST3051 and KAMCLST3152 belonged to the same species. Whole genome analysis of the strain KEBCLARHB70RT and average amino acid identity values confirmed its distinctive phylogenetic position within the family Acetobacteraceae. Phenotypic, ecological and genomic characteristics distinguished strains KEBCLARHB70RT, KAMCLST3051 and KAMCLST3152 from all genera in the family Acetobacteraceae. Therefore, we propose a novel genus and a novel species, Lichenicoccus roseus gen. nov., sp. nov., for these novel Acetobacteraceae members. Strain KEBCLARHB70RT (=KCTC 72321T=VKM B-3305T) has been designated as the type strain.

Potassium usnate, a water-soluble usnic acid salt, shows enhanced activity against Schistosoma mansoni in vitro.

Here, we report enhanced the in vitro effect of potassium usnate on coupled adult Schistosoma mansoni worms at different time intervals and concentrations. The evaluated schistosomicidal parameters were the following: motility, mortality, fecundity and integumentary changes, as viewed in photomicrographs. Potassium usnate was able to cause 100 and 50% mortality at 100 and 50 µM concentrations, respectively, after 24 h of exposure, while 25 and 12.5 µM concentrations caused changes in motility at 48 and 72 h, and lethality at 96 and 120 h respectively. Eggs were not detected at any of the concentrations analyzed. Photomicrographs revealed morphological tegument alterations within all periods of observation, such as swelling, blisters, dorsoventral contraction, short and curved worms. In conclusion, our results indicate that potassium usnate represents a possible candidate for a new drug in the control of schistosomiasis.

A spectral analysis of common boreal ground lichen species.

Lichens dominate a significant part of the Earth's land surface, and are valuable bioindicators of various environmental changes. In the northern hemisphere, the largest lichen biomass is in the woodlands and heathlands of the boreal zone and in tundra. Despite the global coverage of lichens, there has been only limited research on their spectral properties in the context of remote sensing of the environment. In this paper, we report spectral properties of 12 common boreal lichen species. Measurements of reflectance spectra were made in laboratory conditions with a standard spectrometer (350-2500 nm) and a novel mobile hyperspectral camera (400-1000 nm) which was used in a multiangular setting. Our results show that interspecific differences in reflectance spectra were the most pronounced in the ultraviolet and visible spectral range, and that dry samples always had higher reflectance than fresh (moist) samples in the shortwave infrared region. All study species had higher reflectance in the backward scattering direction compared to nadir or forward scattering directions. Our results also reveal, for the first time, that there is large intraspecific variation in reflectance of lichen species. This emphasizes the importance of measuring several replicates of each species when analyzing lichen spectra. In addition, we used the data in a spectral clustering analysis to study the spectral similarity between samples and species, and how these similarities could be linked to different physical traits or phylogenetic closeness of the species. Overall, our results suggest that spectra of some lichen species with large ground coverage can be used for species identification from high spatial resolution remote sensing imagery. On the other hand, for lichen species growing as small assemblages, mobile hyperspectral cameras may offer a solution for in-situ species identification. The spectral library collected in this study is available in the SPECCHIO Spectral Information System.

Hepatoprotective activity of depsidone enriched Cladonia rangiferina extract against alcohol-induced hepatotoxicity targeting cytochrome P450 2E1 induced oxidative damage.

Alcoholic liver disease (ALD) is a broad-spectrum disorder, covering fatty liver, cirrhosis, alcoholic hepatitis and in extreme untreated condition hepatocellular carcinoma (HCC) may also develop. Cladonia rangiferina (CR) is a class of lichen having a broad spectrum of pharmacological activity. It is used like traditional natural sources in ancient times in India, China, Sri Lanka, etc. Folkloric record about CR has reported their use as an antimicrobial, antitumor, antioxidant, anti-inflammatory activities, etc. Hence, the present study was requested to ascertain the effect of the ethanolic extract of Cladonia rangiferina (CRE) on alcohol-induced hepatotoxicity. The animals were evaluated for the estimation of the liver in vivo biochemical antioxidant parameters. The liver tissues were further evaluated histopathologically and western blotting examination for localization of apoptotic gene expression that plays a pivotal role in hepatotoxicity. The results of this study reveal that CRE proves to be helpful in the treatment of alcohol-induced hepatotoxicity and oxidative stress. Results of different markers have shown that among all, CRE has demonstrated the best hepatoprotective activity. These observations say about the importance of the components of the extract. The ameliorative action of CRE in alcoholic liver damage may exist due to antioxidant, anti-inflammatory, and anti-apoptotic activities.

Integrity of lichen cell membranes as an indicator of heavy-metal pollution levels in soil.

The epigeic lichens Cladonia rei and Diploschistes muscorum are effective heavy-metal-tolerant colonisers of highly polluted and disturbed sites. In this study we compare their bioaccumulation capacities, accumulation patterns, and responses to heavy-metal stress, as expressed in terms of cell membrane damage. We also aim at verifying the relationships between cell membrane damage and levels of soil pollution with heavy metals, and thereby to identify the bioindicative value of this physiological parameter. Total and intracellular concentrations of Zn, Pb, Cd, As, Cu, and Ni were measured in 140 samples of lichens and corresponding soil, collected from variously contaminated sites. Relative electrical conductivity (EC%) values were determined concurrently in the lichen samples. The studied lichens differ considerably in intracellular uptake susceptibility and the related reduction in membrane integrity. In C. rei thalli, more than half of Zn, Pb, Cd, and As loads are accumulated extracellularly, whereas D. muscorum exhibits a tendency towards intracellular accumulation of the same elements. This property is clearly reflected in cell membrane damage, which is considerably greater in the latter species irrespective of study site. This indicates that intracellular heavy-metal accumulation affects the level of cell membrane damage. Two soil pollution classes were distinguished for both lichens based on element contents in host-substrate samples. The losses of cell membrane integrity in lichen thalli are related to these classes. EC% values above 16 in C. rei and above 20 in D. muscorum suggest elevated levels of heavy metals in the soil. Consequently, this physiological parameter can serve as an early warning indicator for detection of elevated metal concentrations in soil. The biomonitoring method proposed here involves common and widespread lichen species and can be widely applied in post-industrial areas.

Usnic Acid Potassium Salt: Evaluation of the Acute Toxicity and Antinociceptive Effect in Murine Model.

To obtain usnic acid potassium salt (PS-UA), the usnic acid (UA) was extracted and purified from the lichen Cladonia substellata, and modified to produce PS-UA. The structure was determined by 1H-NMR, IR and elemental analysis, ratified through computational models, as well as identification the site of K+ insertion in the molecule. Antinociceptive activity was detected through contortions in mice induced by acetic acid and formalin (phases I and II) after treatments with 10 and 20 mg/kg of PS-UA, indicating interference in both non-inflammatory and inflammatory pain. After oral administration at doses of 500, 1000 and 2000 mg/kg, no deaths of mice with treatments below 2000 mg/kg were observed. Except for body weight gain, food and water consumption decreased with treatments of 1000 and 2000 mg/kg, and the number of segmented leukocytes was higher for both treatments. Regarding serum levels, cholesterol and triglycerides decreased, however, there was an increase in hepatic transaminases with both treatments. Liver and kidney histological changes were detected in treatments of 2000 mg/kg, while the spleen was preserved. The PS-UA demonstrated antinociceptive activity while the acute toxicity at the concentration of 2000 mg/kg was the only dose that presented morphological changes in the liver and kidney.

Edaphobacter lichenicola sp. nov., a member of the family Acidobacteriaceae from lichen-dominated forested tundra.

An isolate of aerobic, Gram-stain-negative, rod-shaped, non-motile and light-pink pigmented bacteria, designated SBC68T, was obtained from slightly decomposed thalli of the lichen Cladonia sp. collected from the forested tundra of north-western Siberia. Cells of this isolate occurred singly, in pairs or in rosettes. These bacteria were acidophilic (optimum growth at pH 4.3-5.6) and mesophilic (optimum growth at 20-30 °C) but were also capable of growth at low temperatures, down to 7 °C. The preferred growth substrates were sugars, some organic acids and lichenan. The major fatty acids were iso-C15 : 0, C16 : 1ω7c, C16 : 0, C16 : 1ω7t, and 13,16-dimethyl octacosanedioic acid. The only quinone was MK-8, and the G+C content of the DNA was 54.7 mol%. SBC68T represented a member of the family Acidobactericeae; the closest taxonomically described relatives were Edaphobacter dinghuensis DHF9T and Granulicella aggregans TPB6028T (97.2 and 97.1 % 16S rRNA gene sequence similarity, respectively). In 16S rRNA gene-based trees, SBC68T clustered together with species of the genus Edaphobacter. However, this isolate differed from all previously described species of the genus Edaphobacter with respect to the pink pigmentation, formation of cell rosettes and substrate utilization pattern. On the basis of these data, strain SBC68T should be considered to represent a novel species of acidobacteria, for which the name Edaphobacter lichenicola sp. nov. is proposed. The type strain is SBC68T (=DSM 104462T=VKM B-3208T).

The antioxidant and antigenotoxic potential of methanol extract of Cladonia foliacea (Huds.) Willd.

In this article, the genotoxic and antigenotoxic effects of methanol extract of of Cladonia foliacea (Huds.) Willd. (CME) were studied using WP2, Ames (TA1535 and TA1537), and sister chromatid exchange (SCE) test systems. The results of our studies showed that 5 µM concentration of aflatoxin B1(AFB1) changed the frequencies of SCE and malondialdehyde (MDA) levels, superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx) activities. When 5 and 10 µg/mL concentrations of CME was added to AFB1, the frequencies of SCE and MDA level were decreased and SOD, GSH, and GPx levels were increased. The extract CME did not show any mutagenicity on Ames (Salmonella typhimurium TA1535, TA1537) and WP2 (Escherichia coli) test systems. On the other hand, CME has antimutagenicity on the mentioned test systems. The results of this experiment have clearly shown that CME has a significant antioxidative and antigenotoxic effect, which is thought to be due to the antigenotoxic activities of antioxidant enzymes.

Algal and fungal diversity in Antarctic lichens.

The composition of lichen ecosystems except mycobiont and photobiont has not been evaluated intensively. In addition, recent studies to identify algal genotypes have raised questions about the specific relationship between mycobiont and photobiont. In the current study, we analyzed algal and fungal community structures in lichen species from King George Island, Antarctica, by pyrosequencing of eukaryotic large subunit (LSU) and algal internal transcribed spacer (ITS) domains of the nuclear rRNA gene. The sequencing results of LSU and ITS regions indicated that each lichen thallus contained diverse algal species. The major algal operational taxonomic unit (OTU) defined at a 99% similarity cutoff of LSU sequences accounted for 78.7-100% of the total algal community in each sample. In several cases, the major OTUs defined by LSU sequences were represented by two closely related OTUs defined by 98% sequence similarity of ITS domain. The results of LSU sequences indicated that lichen-associated fungi belonged to the Arthoniomycetes, Eurotiomycetes, Lecanoromycetes, Leotiomycetes, and Sordariomycetes of the Ascomycota, and Tremellomycetes and Cystobasidiomycetes of the Basidiomycota. The composition of major photobiont species and lichen-associated fungal community were mostly related to the mycobiont species. The contribution of growth forms or substrates on composition of photobiont and lichen-associated fungi was not evident.

Expectorant and antioxidant activities of purified fumarprotocetraric acid from Cladonia verticillaris lichen in mice.

UNLABELLED: The lichen Cladonia verticillaris produces bioactive secondary metabolites, such as fumarprotocetraric (FUM) and protocetraric acids. Species of the genus Cladonia demonstrate anti-tumor, anti-inflammatory and antipyretic activities and have been used in folk medicine to treat respiratory diseases (throat irritation, cough, asthma and tuberculosis). The aim of the present study was to evaluate the expectorant and mucolytic activities of fumarprotocetraric acid in albino Swiss mice. FUM was extracted and purified from an acetone extract of C. verticillaris. The phenol red quantification method was used on the bronchoalveolar lavage fluid following the administration of FUM (25, 50 or 100 mg/kg orally or intraduodenally and 12.5, 25 or 50 mg/kg, intraperitoneally) for the evaluation of expectorant activity. Control groups received either saline solution (7.5 mL/kg) or ambroxol (1 mg/kg) through the same administration routes. Antioxidant activity was evaluated using the thiobarbituric acid reactive species assay in mouse lung tissue treated with the FUM at 25, 50 or 100 mg/kg orally, followed by a lipopolysaccharide solution at 1 mg/kg intrapleurally. The same protocol was used for the control groups using either saline solution (7.5 mL/kg, orally) or N-acetylcysteine (20 mg/kg, orally). RESULTS: Orally administered FUM at doses of 25 and 50 mg/kg promoted significantly greater dose-dependent phenol red activity in the bronchoalveolar lavage and expectorant activity in comparison to the controls (p < 0.05). Lipid peroxidation (malondialdehyde equivalent) was reduced by 50% in the lung tissue. CONCLUSION: The results confirm the expectorant and antioxidant properties of fumarprotocetraric acid produced by the lichen C. verticillaris.

Influence of prescribed burning on reindeer winter pastures at landscape scale in northern Sweden: A modelling approach.

Whilst the re-introduction of fire can contribute to biodiversity conservation in Fennoscandian forests, the effects on reindeer herding remain uncertain. To assess the short- and long-term effects of prescribed burning on lichen supply in a productive forest landscape, we developed a model simulating lichen biomass available for reindeer grazing, covering 300 years and 1500 pine stands, under different soil preparation scenarios, including different prescribed burning regimes and mechanical scarification. Our simulations revealed that burning 25-50% of yearly clear-cuts has the potential to stop, or even reverse, reindeer lichen decline at landscape scale after 70 years, greatly surpassing the short-term losses caused by burning. No burning or burning 5% of yearly clear-cuts, as required by the FSC certification, compounded the negative effects of fire suppression and scarification on lichen. Compared to the scenario with no soil preparation, all our simulations resulted in a continuous decrease of lichen supply in Lichen-type stands, indicating that any form of disturbance in these habitats can strongly limit future gains.

Myrmecia, Not Asterochloris, Is the Main Photobiont of Cladonia subturgida (Cladoniaceae, Lecanoromycetes).

This study explores the diversity of photobionts associated with the Mediterranean lichen-forming fungus Cladonia subturgida. For this purpose, we sequenced the whole ITS rDNA region by Sanger using a metabarcoding method for ITS2. A total of 41 specimens from Greece, Italy, France, Portugal, and Spain were studied. Additionally, two specimens from Spain were used to generate four cultures. Our molecular studies showed that the genus Myrmecia is the main photobiont of C. subturgida throughout its geographic distribution. This result contrasts with previous studies, which indicated that the main photobiont for most Cladonia species is Asterochloris. The identity of Myrmecia was also confirmed by ultrastructural studies of photobionts within the lichen thalli and cultures. Photobiont cells showed a parietal chloroplast lacking a pyrenoid, which characterizes the species in this genus. Phylogenetic analyses indicate hidden diversity within this genus. The results of amplicon sequencing showed the presence of multiple ASVs in 58.3% of the specimens studied.

An Exception to the Rule? Could Photobiont Identity Be a Better Predictor of Lichen Phenotype than Mycobiont Identity?

With rare exceptions, the shape and appearance of lichen thalli are determined by the fungal partner; thus, mycobiont identity is normally used for lichen identification. However, it has repeatedly been shown in recent decades that phenotypic data often does not correspond with fungal gene evolution. Here, we report such a case in a three-species complex of red-fruited Cladonia lichens, two of which clearly differ morphologically, chemically, ecologically and in distribution range. We analysed 64 specimens of C. bellidiflora, C. polydactyla and C. umbricola, mainly collected in Europe, using five variable mycobiont-specific and two photobiont-specific molecular markers. All mycobiont markers exhibited very low variability and failed to separate the species. In comparison, photobiont identity corresponded better with lichen phenotype and separated esorediate C. bellidiflora from the two sorediate taxa. These results can be interpreted either as an unusual case of lichen photomorphs or as an example of recent speciation, in which phenotypic differentiation precedes the separation of the molecular markers. We hypothesise that association with different photobionts, which is probably related to habitat differentiation, may have triggered speciation in the mycobiont species.

Bioassay-guided isolation of antimycobacterial substances from the traditionally used lichen Cladonia pyxidata (L.) Hoffm.

The aim of the present study is to provide a scientific rationale for the folklore usage of Cladonia pyxidata (L.) Hoffm. in treating tuberculosis (Tb). Through bioassay-guided isolation, antimycobacterial metabolites were isolated from under-investigated lichen C. pyxidata and examined against M.t H37Ra and six MDR strains. Further, the cytotoxicity of all isolated metabolites was evaluated on THP-1 macrophages. Bioassay-guided isolation of acetone extract of C. pyxidata yielded four metabolites, namely usnic acid, atranorin, barbatic acid, and fumarprotocetraric acid. Among those, the MIC values of usnic acid and fumarprotocetraric acid showed more effective in inhibiting the growth of six MDR strains, compared to first-line drug rifampicin. In addition, the 50% inhibitory concentration values of these two compounds on THP-1 were found to be far higher than MIC values against tested Tb strains, indicating that THP-1 macrophages were not harmfully affected at concentrations that were effective against M.t and MDR strains. The results exposed the traditional use of C. pyxidata for treating Tb, and the key metabolites were found to be usnic acid and fumarprotocetraric acid. The current study lends the first evidence for the presence of antimycobacterial compounds in C. pyxidata. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03159-6.

UHPLC-MS Chemical Fingerprinting and Antioxidant, Enzyme Inhibition, Anti-Inflammatory In Silico and Cytoprotective Activities of Cladonia chlorophaea and C. gracilis (Cladoniaceae) from Antarctica.

The lichen species Cladonia chlorophaea and C. gracilis (Cladoniaceae) are widely distributed in the island archipelago of maritime Antarctica and represent a natural resource of scientific interest. In this work, the metabolomic characterization of the ethanolic extracts of these species and the determination of the antioxidant activity, enzymatic inhibition and anti-inflammatory potential of selected compounds on the 5-lipoxygenase enzyme by molecular docking and cytoprotective activity in the SH-SY5Y cell line were carried out. Nineteen compounds were identified by liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS) in each of the species. The contents of phenolic compounds, antioxidant activity, the inhibition of cholinesterases (acetylcholinesterase and butyrylcholinesterase) and digestive enzymes (α-glucosidase and pancreatic lipase) were variable among species, with better results in C. chlorophaea. Molecular docking evidenced significant binding affinities of some compounds for the 5-lipoxygenase enzyme, together with outstanding pharmacokinetic properties. Both extracts were shown to promote cell viability and a reduction in reactive oxygen species production in an H2O2-induced oxidative stress model. This study contributes to the chemical knowledge of the Cladonia species and demonstrates the biological potential for the prevention and promising treatment of central nervous system pathologies, inflammatory disorders and metabolic alterations.