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In this study, isolation and purification of lichen substances from Usnea cornuta were performed using conventional solvents, green solvents and green technologies. In addition, several lichen compounds were tentatively identified by UHPLC/ESI/MS/MS and usnic acid, diffractaic and galbinic acids were quantified as well. Limonene, ethyl lactate and methanol, were compared regarding their extraction properties and antioxidant capacities, determined by DPPH, ORAC, and FRAP assays. In the ethyl lactate, methanol and limonene extracts, 28 compounds in all, were detected for the first time by high resolution UHPLC-MS/MS fingerprinting. Untargeted metabolomics tentatively identified 14 compounds from the methanolic extract, 4 from limonene extract, and 20 metabolites from ethyl lactate extract. The green extract of ethyl lactate showed a similar antioxidant capacity to toxic methanol extract, except at ORAC assay where it was higher. Therefore, ethyl lactate can replace methanol, to provide more sustainable green chemistry methods.

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Antarctica has a great diversity of microorganisms with biotechnological potential but is not very well Known about yeasts with phosphate solubilization activity. Thus, the aim of this study was to evaluate the ability of yeasts from Antarctica lichens to solubilize phosphate in vitro. In the screening, 147 yeasts were tested and 43 (29%) showed P solubilization in solid NBRIP medium at 15.0 °C, with a higher prevalence of positive genera Vishniacozyma, followed by Cystobasidium. Most of the positive yeasts were isolated from Usnea auratiacoatra, followed by Polycauliona regalis and Lecania brialmontii. Two strains with better activity after screening were selected for the solubilization in the liquid medium, Vishniacozyma victoriae 2.L15 and A.L6 (unidentified). Vishniacozyma victoriae 2.L15 exhibiting activities at 25.0 °C (29.91 mg/L of phosphate and pH 6.85) and at 30.0 °C (619.04 mg/L of phosphate and pH 3.73) and A.L6 strain at 25.0 °C (25.05 mg/L of phosphate and pH 6.69) and at 30.0 °C (31.25 mg/L of phosphate and pH 6.47). Of eight organic acids tested by HPLC, tartaric and acetic acids were detected during phosphate solubilization, with greater release in the period of 144 (2.13 mg/L) and 72 (13.72 mg/L) hours, respectively. Future studies to elucidate the presence of functional genes for P metabolism in lichens, as well as studies in the field of proteomics for the discovery of yeast proteins related to P solubilization are needed. Thus, the high prevalence of lichen-associated yeast communities probably contributed to the high frequency of phosphate-solubilizing isolates in this study.

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The deposition of trace elements around a pulp and paper industry in Morelia, Mexico, was evaluated using two lichen species as biomonitors. Samples of the foliose lichen Flavopunctelia praesignis and the fruticose lichen Usnea ceratina were collected in two remote areas and transplanted at different distances and directions from the pollution source. Lichen samples were exposed for 4 months (1) around the industrial area and (2) in their native habitats (control sites). We investigated the bioaccumulation of 11 trace elements in lichen thalli, and we compared the response of the two lichen species. To identify possible common sources, we evaluated the relationships between trace elements by correlations and cluster analyses. Our results showed that Cd was a good tracer for air pollution from the pulp and paper mills. In samples of Usnea ceratina exposed around the industrial area, Cd was significantly higher than in the remote area. Within the study area, trace element contents increase with the distance from the source, and they showed high depositions in the direction of prevailing winds. Moreover, we were able to detect groups of elements with similar behavior and common origins. Our results indicated that Flavopunctelia praesignis showed a higher capacity to accumulate trace elements than Usnea ceratina.

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Resumen Los líquenes son asociaciones simbióticas mutualistas que se dan entre un hongo y organismos fotoautótrofos tales como algas o cianobacterias. Presentan altas concentraciones de sustancias liquénicas, las cuales son químicamente complejas y han presentado una marcada actividad antifúngica y antibacteriana. Objetivo: Evaluar la actividad antifúngica y antibacteriana (in vitro) del extracto etanólico de Usnea laevis frente a Candida albicans, Staphylococcus aureus y Pseudomonas aeruginosa. Material y métodos: Las sustancias liquénicas presentes en Usnea laevis fueron extraídas mediante el método de Soxhlet y fueron analizadas químicamente por cromatografía acoplado a espectrometría de masas. Para la evaluación de la actividad antifúngica y antibacteriana se utilizó la técnica de Kirby-Bauer. Resultados: Dentro de la composición química del extracto liquénico se tuvo ácido úsnico, eugenol, ergosterol entre otros. Para una concentración de 27 mg/ml del extracto etanólico de Usnea laevis, Staphylococcus aureus fue encontrado sensible siendo esta concentración la que mejores resultados de inhibición arrojó; mientras que la concentración de 11 mg/ml no fue efectiva frente a las cepas evaluadas. Conclusiones: El extracto etanólico de Usnea laevis posee propiedades antifúngicas y antibacterianas frente a Candida albicans, Pseudomonas aeruginosa y Staphylococcus aureus.

Summary Lichens are symbiotic associations between fungi and seaweed or cyanobacteria. Objective: to evaluate the in-vitro antifungal and antibacterial activity of the ethanolic extract of Usnea laevis against Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa. Methods: lichened substances present in Usnea laevis were extracted using the Soxhlet methods and were analyzed by chromatography coupled to mass spectrometry. The Kirby-Bauer method was used to evaluate both the antibacterial and antifungal activity. Results: Usnic acid, eugenol, and ergosterol were the compounds found. S. aureus was sensitive at a concentration of 27 mg/ml of the enolic extract, the 11 mg/ml concentration was not effective. Conclusions: The ethanolic extract of Usnea laevis has antibacterial and antifungal properties against the pathogens tested.

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Purpose:To investigate the effects of the EtOAc extract of U. longissima which is uninvestigated previously on esophagogastric cancer induced in rats with N-methyl-N-nitro-N-nitrosoguanidin (MNNG).Methods:The anticancer activity of EtOAc extract of U. longissima was examined in the esophagogastric adenocarcinoma models induced in rats with MNNG. EtOAc extract of U. longissima, 50 and 100 mg/kg oral doses were administered once daily for six months. MNNG induced differentiated and undifferentiated type adenocarcinomas in the esophageal and gastric tissues of rats.Results:EtOAc extract of U. longissima obtained from U. longissima prevented gastric and esophageal cancerogenesis induced in rats with MNNG. EtOAc extract of U. longissima did not have a lethal effect at doses of 500, 1000 and 2000 mg/kg. The prominent anticarcinogenic activity of EtOAc extract of U. longissima 50 and 100 mg/kg suggests that it is not toxic and it is selective to the cancer tissue.Conclusion:This information may shed light on clinical implementation of EtOAc extract of U. longissima in future.(AU)

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Abstract Purpose: To investigate the effects of the EtOAc extract of U. longissima which is uninvestigated previously on esophagogastric cancer induced in rats with N-methyl-N-nitro-N-nitrosoguanidin (MNNG). Methods: The anticancer activity of EtOAc extract of U. longissima was examined in the esophagogastric adenocarcinoma models induced in rats with MNNG. EtOAc extract of U. longissima, 50 and 100 mg/kg oral doses were administered once daily for six months. MNNG induced differentiated and undifferentiated type adenocarcinomas in the esophageal and gastric tissues of rats. Results: EtOAc extract of U. longissima obtained from U. longissima prevented gastric and esophageal cancerogenesis induced in rats with MNNG. EtOAc extract of U. longissima did not have a lethal effect at doses of 500, 1000 and 2000 mg/kg. The prominent anticarcinogenic activity of EtOAc extract of U. longissima 50 and 100 mg/kg suggests that it is not toxic and it is selective to the cancer tissue. Conclusion: This information may shed light on clinical implementation of EtOAc extract of U. longissima in future.

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Los líquenes son producto de una asociación simbiótica entre un hongo y alga y/o cianobacteria; esta simbiosis produce una serie de metabolitos secundarios o sustancias liquénicas únicas, las mismas que son aisladas a partir de sus extractos y que han presentado una marcada actividad antibiótica y antifúngica. Considerando que en el Ecuador no se tienen antecedentes acerca de este tipo de estudios, el objetivo planteado fue el evaluar la actividad antifúngica del extracto liquénico de Usnea laevis frente a hongos fitopatógenos. En el presente estudio las sustancias liquénicas almacenadas en el talo de Usnea laevis fueron extraídas con metanol y etanol. La actividad antifúngica in vitro de estos extractos fue probado frente a géneros de hongos fitopatógenos previamente aislados (Aspergillus, Penicillium y Rhizopus stolonifer); añadiéndolo al medio de cultivo Agar Papa Dextrosa (PDA) a una concentración de 0,5%; determinándose el porcentaje de inhibición. Los datos fueron analizados estadísticamente y se realizó la clasificación del extracto en base a su porcentaje de inhibición según la OILB. El extracto metanólico y etanólico de Usnea laevis mostró una mayor efectividad frente a Rhizopus stolonifer, inhibiendo más del 50% de su crecimiento, a diferencia de Penicillium y Aspergillus cuyo porcentaje de inhibición fue mucho menor y mostraron diferencias estadísticamente significativas. El extracto liquénico fue moderadamente tóxico para Rhizopus stolonifer, ligeramente tóxico para Aspergillus sp 1 y Penicillium sp e inocuo para Aspergillus sp 2.

A lichen is an organism product of a symbiotic association between a fungus and algae and / or cyanobacteria; this symbiosis produces many secondary metabolites or unique lichen substances, which are isolated from their extracts and show a marked antibiotic and antifungal activity. Considering there is no background on this type of studies in Ecuador, the aim of this study was to evaluate the antifungal in vitro activity of Usnea laevis extract on phytopathogenic fungi. The lichenic substances stored in the thallus of Usnea laevis were extracted with methanol and ethanol. The in vitro antifungal activity of these extracts was tested against phytopathogenic fungal genera previously isolated (Aspergillus, Penicillium and Rhizopus stolonifer) . They were adding to Potato Dextrose Agar (PDA) culture medium at a concentration of 0.5%, and the inhibition percentage was determined. Data were statistically analyzed and the extract was classified based on its inhibition percentage according to the OILB. Methanolic and ethanolic extracts of Usnea laevis showed greater effectiveness against Rhizopus stolonifer, inhibiting more than 50% of its growth, unlike Penicillium and Aspergillus, whose inhibition percentage was lower and showed significant differences. The liquid extract was also moderately toxic to Rhizopus stolonifer, slightly toxic to Aspergillus sp 1 and Penicillium sp and harmless to Aspergillus sp 2.

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Lichens are symbiotic associations of fungi with microalgae and/or cyanobacteria, which are considered among the slowest growing organisms, with strong tolerance to adverse environmental conditions. There are about 400 genera and 1600 species of lichens and those belonging to the Usnea genus comprise about 360 of these species. Usnea lichens have been used since ancient times as dyes, cosmetics, preservatives, deodorants and folk medicines. The phytochemistry of the Usnea genus includes more than 60 compounds which belong to the following classes: depsides, depsidones, depsones, lactones, quinones, phenolics, polysaccharides, fatty acids and dibenzofurans. Due to scarce knowledge of metabolomic profiles of Usnea species (U. barbata, U. antarctica, U. rubicunda and U. subfloridana), a study based on UHPLC-ESI-OT-MS-MS was performed for a comprehensive characterization of their secondary metabolites. From the methanolic extracts of these species a total of 73 metabolites were identified for the first time using this hyphenated technique, including 34 compounds in U. barbata, 21 in U. antarctica, 38 in U. rubicunda and 37 in U. subfloridana. Besides, a total of 13 metabolites were not identified and reported so far, and could be new according to our data analysis. This study showed that this hyphenated technique is rapid, effective and accurate for phytochemical identification of lichen metabolites and the data collected could be useful for chemotaxonomic studies.

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ABSTRACT The larvicidal activity of the methanol extract, fractions and compounds 2-hydroxy-4-methoxy-6-propyl-methyl benzoate and (+)-usnic acid identified from the lichen Ramalina usnea (L.) R. Howe, Ramalinaceae, was tested against the third instar larvae of the Aedes aegypti mosquito. The methanol extract and three fractions showed activity, killing 100% and 96.6% of the larvae at a concentration of 150 µg/ml at 24 h. The isolated compounds, 2-hydroxy-4-methoxy-6-propyl-methyl benzoate and the (+)-usnic acid showed larvicidal activity, presenting LC50 values of 4.85 and 4.48 µg/ml, respectively. This is the first study of its kind reporting the larvicidal activity against the A. aegypti mosquito with compound (1).

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Lichen Usnea barbata transplants were tested as a biomonitor of atmospheric deposition in an apparently pristine environment that is Tierra del Fuego region (Patagonia, Argentina). The present survey is connected with the volcanic eruption that started in north Patagonia on June 4, 2011 from the Puyehue-Cordón Caulle volcano, Chile (north Patagonia, at 1700 km of distance of our sampling sites). Lichens were collected in September 2011 (one month of exposure) and September 2012 (1 year of exposure) in 27 sites covering the northern region of the province where trees are not present. The atmospheric deposition of 27 elements by using Neutron Activation Analysis (NAA) was determined in the collected samples. The first aim of the study was to evaluate the influence of the volcanic eruption on the regional atmospheric deposition comparing our results with baseline data we determined in U. barbata in 2006 in the same sites. The second aim was to test possible patterns of bioaccumulation between the two sampling campaigns after the volcanic eruption. With respect to 2006 baseline levels, we found significant higher levels for As, Ba, Co, Cr, Cs, Na, Sb and U in lichens collected after 1 month of exposure (first sampling campaign--2011). Between the two sampling campaigns (2011-2012) after the eruption, lichens reflected the natural contamination by volcanic ashes with significantly higher median levels of Br, Cr, Fe, K, Na, Sc, and Se. Results confirmed the very good aptitude of U. barbata to reflect the levels of elements in the environment at global scale and to reflect the volcanic emissions at distant places. Volcanic eruptions cause the emission in the atmosphere of elevated levels of particulate matter. In this regard, our findings demonstrate the importance to evaluate the metal composition of the particles to avoid possible health effects.

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