Cytotoxic, anti-proliferative, and apoptotic evaluation of Ramalina sinensis (Ascomycota, Lecanoromycetes), lichenized fungus on oral squamous cell carcinoma cell line; in-vitro study.

BACKGROUND: Scientists and medical professionals are actively striving to improve the efficacy of treatment methods for oral squamous cell carcinoma (OSCC), the most frequently occurring cancer within the oral cavity, by exploring the potential of natural products. The active pharmacological compounds found in lichenized fungi have shown potential for aiding in cancer treatment. Recent research aims to evaluate the impact of the lichenized fungus Ramalina sinensis (R. sinensis) on the cell viability and apoptosis of OSCC cell lines, considering the anti-inflammatory and anti-cancer capabilities of lichens. METHODS: Ramalina sinensis (Ascomycota, Lecanoromycetes) was selected for investigation of its effects on a human oral squamous cell carcinoma cell line. Acetone and methanol extracts of R. sinensis on an OSCC cell line (KB cell line, NCBI Code: C152) were investigated. Viability was assessed by MTT assay analysis, and apoptotic cells were measured using flow cytometry analysis. Scratch assay was used to assess cell migration. The chemical composition and metabolic profiling of R. sinensis were investigated. RESULTS: The growth and multiplication of KB cells were observed to undergo a gradual but remarkable inhibition when exposed to various concentrations. Specifically, concentrations of 6.25, 12.5, 25, 50, 100, and 200 µg/mL exhibited a significant suppressive effect on the proliferation of KB cells. The inhibition of cell proliferation exhibited a statistically significant difference between the extracts obtained from acetone and methanol. Flow cytometry results show an increase in apoptosis of OSCC cells by acetone extract. R. sinensis exerted a concentration-dependent inhibitory effect on the migration of OSCC cells. The chemical composition of R. sinensis was investigated using liquid chromatography positive ion electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), and 33 compounds in the acetone and methanol extracts of R. sinensis were detected. CONCLUSION: The findings provide evidence supporting the beneficial effects of R. sinensis extract on inducing apoptosis in OSCC cells and exerting anti-cancer properties.

Metabarcoding of Antarctic Lichens from Areas with Different Deglaciation Times Reveals a High Diversity of Lichen-Associated Communities.

Lichens have developed numerous adaptations to optimise their survival under harsh abiotic stress, colonise different substrates, and reach substantial population sizes and high coverage in ice-free Antarctic areas, benefiting from a symbiotic lifestyle. As lichen thalli represent consortia with an unknown number of participants, it is important to know about the accessory organisms and their relationships with various environmental conditions. To this end, we analysed lichen-associated communities from Himantormia lugubris, Placopsis antarctica, P. contortuplicata, and Ramalina terebrata, collected from soils with differing deglaciation times, using a metabarcoding approach. In general, many more Ascomycete taxa are associated with the investigated lichens compared to Basidiomycota. Given our sampling, a consistently higher number of lichen-associated eukaryotes are estimated to be present in areas with deglaciation times of longer than 5000 years compared to more recently deglaciated areas. Thus far, members of Dothideomycetes, Leotiomycetes, and Arthoniomycetes have been restricted to the Placopsis specimens from areas with deglaciation times longer than 5000 years. Striking differences between the associated organisms of R. terebrata and H. lugubris have also been discovered. Thus, a species-specific basidiomycete, Tremella, was revealed for R. terebrata, as was a member of Capnodiales for H. lugubris. Our study provides further understanding of the complex terricolous lichen-associated mycobiome using the metabarcoding approach. It also illustrates the necessity to extend our knowledge of complex lichen symbiosis and further improve the coverage of microbial eukaryotes in DNA barcode libraries, including more extended sampling.

Analyzing the Impact of Ramalina digitellata, R. fastigiata, R. fraxinea, and R. polymorpha's Usnic Acid Concentration on Antioxidant, DNA-Protective, Antimicrobial, and Cytotoxic Properties.

The present study is focused on the antimicrobial, antioxidant, cytotoxic, and DNA protective effects of methanol extract obtained from R. digitellata, R. fastigiata, R. fraxinea, and R. polymorpha species that are distributed in Turkey. The highest total phenol content was determined in R. digitellata (144.6 mgGAE/gextract ), and the highest total amount of flavonoids was found in R. fastigiata (20.40 mgGAE/gextract ). The content of usnic acid was determined by High-Performance Liquid Chromatography (HPLC) and the highest amount was found in R. digitellata. DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS [2,2'-azinobis(3-ethylbenzathiazoline-6-sulfonic acid)] radical scavenging methods were used for antioxidant activity. R. fraxinea showed the highest DPPH⋅ and ABTS+ ⋅ scavenging activity. In addition, the DNA protective effect was investigated using pBR322 plasmid DNA, and; all studied species were found to have DNA protective effects. The antibacterial activity was investigated using the disc diffusion method, and the R. digitellata methanol extract showed the best results with a 12.35 mm zone on Proteus mirabilis. On the human lung cancer (A549) and breast cancer (MDA-MB-231) cell lines, cytotoxic activity was assessed using an MTT assay. All lichen extracts were found to have a significant cytotoxic effect on both cancer cell lines at 1000 µg/mL concentration. These results suggest that Ramalina species may be potential candidates for developing new phytopharmaceuticals and functional components.

Near-Infrared Metabolomic Fingerprinting Study of Lichen Thalli and Phycobionts in Culture: Aquaphotomics of Trebouxia lynnae Dehydration.

Near-infrared spectroscopy (NIRS) is an accurate, fast and safe technique whose full potential remains to be exploited. Lichens are a paradigm of symbiotic association, with extraordinary properties, such as abiotic stress tolerance and adaptation to anhydrobiosis, but subjacent mechanisms await elucidation. Our aim is characterizing the metabolomic NIRS fingerprints of Ramalina farinacea and Lobarina scrobiculata thalli, and of the cultured phycobionts Trebouxia lynnae and Trebouxia jamesii. Thalli collected in an air-dry state and fresh cultivated phycobionts were directly used for spectra acquisition in reflectance mode. Thalli water peaks were associated to the solvation shell (1354 nm) and sugar-water interactions (1438 nm). While northern-southern orientation related with two hydrogen bonded (S2) water, the site was related to one hydrogen bonded (S1). Water, lipids (saturated and unsaturated), and polyols/glucides contributed to the profiles of lichen thalli and microalgae. R. farinacea, with higher desiccation tolerance, shows higher S2 water than L. scrobiculata. In contrast, fresh phycobionts are dominated by free water. Whereas T. jamesii shows higher solvation water content, T. lynnae possesses more unsaturated lipids. Aquaphotomics demonstrates the involvement of strongly hydrogen bonded water conformations, polyols/glucides, and unsaturated/saturated fatty acids in the dehydration process, and supports a "rubbery" state allowing enzymatic activity during anhydrobiosis.

Antibiotic and Nematocidal Metabolites from Two Lichen Species Collected on the Island of Lampedusa (Sicily).

The antibiotic and nematocidal activities of extracts from two coastal lichen species collected on Lampedusa Island (Sicily), Ramalina implexa Nyl. and Roccella phycopsis Ach., were tested. Methyl orsellinate, orcinol, (+)-montagnetol, and for the first time 4-chlororcinol were isolated from Roccella phycopsis. (+)-Usnic acid was obtained from Ramalina implexa. The crude organic extract of both lichen species showed strong antibiotic activity against some bacterial species and nematocidal activity. Among all the pure metabolites tested against the infective juveniles (J2) of the root-knot nematode (RKN) Meloydogine incognita, (+)-usnic acid, orcinol, and (+)-montagnetol had significant nematocidal activity, comparable with that of the commercial nematocide Velum® Prime, and thus they showed potential application in agriculture as a biopesticide. On the contrary, methyl orsellinate and 4-chlororcinol had no nematocidal effect. These results suggest that the substituent pattern at ortho-para-position in respect to both hydroxyl groups of resorcine moiety, which is present in all metabolites, seems very important for nematocidal activity. The organic extracts of both lichens were also tested against some Gram-positive and Gram-negative bacteria. Both extracts were active against Gram-positive species. The extract of Ramalina implexa showed, among Gram-negative species, activity against Escherichia coli and Acinetobacter baumannii, while that from Roccella phycopsis was effective towards all test strains, with the exception of Pseudomonas aeruginosa. The antimicrobial activity of (+)-usnic acid, methyl orsellinate, and (+)-montagnetol is already known, so tests were focused on orcinol and 4-chlororcinol. The former showed antibacterial activity against all Gram positive and Gram-negative test strains, with the exception of A. baumannii and K. pneumoniae, while the latter exhibited a potent antibacterial activity against Gram-positive test strains and among Gram-negative strains, was effective against A. baumannii and K. pneumonia. These results suggest, for orcinol and 4-chlororcinol, an interesting antibiotic potential against both Gram-positive and Gram-negative bacterial strains.

Trebouxia lynnae sp. nov. (Former Trebouxia sp. TR9): Biology and Biogeography of an Epitome Lichen Symbiotic Microalga

Two microalgal species, Trebouxia jamesii and Trebouxia sp. TR9, were detected as the main photobionts coexisting in the thalli of the lichen Ramalina farinacea. Trebouxia sp. TR9 emerged as a new taxon in lichen symbioses and was successfully isolated and propagated in in vitro culture and thoroughly investigated. Several years of research have confirmed the taxon Trebouxia sp. TR9 to be a model/reference organism for studying mycobiont−photobiont association patterns in lichen symbioses. Trebouxia sp. TR9 is the first symbiotic, lichen-forming microalga for which an exhaustive characterization of cellular ultrastructure, physiological traits, genetic and genomic diversity is available. The cellular ultrastructure was studied by light, electron and confocal microscopy; physiological traits were studied as responses to different abiotic stresses. The genetic diversity was previously analyzed at both the nuclear and organelle levels by using chloroplast, mitochondrial, and nuclear genome data, and a multiplicity of phylogenetic analyses were carried out to study its intraspecific diversity at a biogeographical level and its specificity association patterns with the mycobiont. Here, Trebouxia sp. TR9 is formally described by applying an integrative taxonomic approach and is presented to science as Trebouxia lynnae, in honor of Lynn Margulis, who was the primary modern proponent for the significance of symbiosis in evolution. The complete set of analyses that were carried out for its characterization is provided.

Novel methods to characterise spatial distribution and enantiomeric composition of usnic acids in four Icelandic lichens.

Usnic acid is an antibiotic metabolite produced by a wide variety of lichenized fungal lineages. The enantiomers of usnic acid have been shown to display contrasting bioactivities, and hence it is important to determine their spatial distribution, amounts and enantiomeric ratios in lichens to understand their roles in nature and grasp their pharmaceutical potential. The overall aim of the study was to characterise the spatial distribution of the predominant usnic acid enantiomer in lichens by combining spatial imaging and chiral chromatography. Specifically, separation and quantification of usnic acid enantiomers in four common lichens in Iceland was performed using a validated chiral chromatographic method. Molecular dynamics simulation was carried out to rationalize the chiral separation mechanism. Spatial distribution of usnic acid in the lichen thallus cross-sections were analysed using Desorption Electrospray Ionization-Imaging Mass Spectrometry (DESI-IMS) and fluorescence microscopy. DESI-IMS confirmed usnic acid as a cortical compound, and revealed that usnic acid can be more concentrated around the algal vicinity. Fluorescence microscopy complemented DESI-IMS by providing more detailed distribution information. By combining results from spatial imaging and chiral separation, we were able to visualize the distribution of the predominant usnic acid enantiomer in lichen cross-sections: (+)-usnic acid in Cladonia arbuscula and Ramalina siliquosa, and (-)-usnic acid in Alectoria ochroleuca and Flavocetraria nivalis. This study provides an analytical foundation for future environmental and functional studies of usnic acid enantiomers in lichens.

Stereocalpin B, a New Cyclic Depsipeptide from the Antarctic Lichen Ramalina terebrata.

Stereocalpin B, a new cyclic depsipeptide (1), and a new dibenzofuran derivative (3), were isolated from the Antarctic lichen, Ramalina terebrata (Ramalinaceae), along with a known cyclic depsipeptide (2). The structures of new compounds were characterized by comprehensive spectrometric analyses; high-resolution fast atom bombardment mass spectrometry (HR-FABMS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Stereocalpin B (1) existed in a rotameric equilibrium, which was confirmed using nuclear Overhauser effect spectroscopy (NOESY)/exchange spectroscopy (EXSY) spectrum. Absolute configurations of the amino acid units in 1 were assigned using the advanced Marfey's method and subsequent NOESY analysis of the 5-hydroxy-2,4-dimethyl-3-oxo-decanoic acid residue confirmed the complete stereochemistry of 1. Compounds 1-3 exhibited moderate antimicrobial activities against E. coli, with the IC50 values ranging from 18-30 µg/mL. Compound 2 exhibited cell growth inhibition against HCT116 cell lines, with the IC50 value of 20 ± 1.20 µM, and compounds 1 and 2 also showed potent anti-inflammatory activities against lipopolysaccharide (LPS)-induced RAW264.7 macrophages with the IC50 values ranging from 5-7 µM.

Three new phenolic compounds from the lichen Ramalina peruviana Ach. (Ramalinaceae).

Three new phenolic compounds, peruvinides A-C were isolated from the lichen Ramalina peruviana Ach. (Ramalinaceae). Their structures were unambiguously determined by extensive spectroscopic analyses and comparison with literature data. Peruvinides A and B bearing unusual moieties were found for the first time among lichen metabolites.

Phytochemical constituents, antioxidant and antistaphylococcal activities of Evernia prunastri (L.) Ach., Pseudevernia furfuracea (L.) Zopf. and Ramalina farinacea (L.) Ach. from Morocco.

The purpose of this work was to assess chemical composition, antibacterial activity against Staphylococcus aureus isolates from catheter-associated infections and antioxidant activity of methanol extracts of three lichens collected from Morocco. The phytochemical analysis of the methanol extracts of these lichens was performed by HPLC-UV method, the predominant phenolic compounds were evernic acid, physodalic acid and usnic acid for Evernia prunastri, Pseudevernia furfuracea and Ramalina farinacea, respectively. Total phenolic compounds and total flavonoid content of all extracts were also determined. As a result, Pseudevernia furfuracea extract had the strongest effect and the highest phenolic compounds content. All extracts showed antibacterial activity against all tested strains (MIC values ranging from 0.078 to 0.625 mg/mL), the strongest inhibition was obtained with the extract of Evernia prunastri.

The Role of Photobionts as Drivers of Diversification in an Island Radiation of Lichen-Forming Fungi.

Speciation in oceanic islands has attracted the interest of scientists since the 19th century. One of the most striking evolutionary phenomena that can be studied in islands is adaptive radiation, that is, when a lineage gives rise to different species by means of ecological speciation. Some of the best-known examples of adaptive radiation are charismatic organisms like the Darwin finches of the Galapagos and the cichlid fishes of the great African lakes. In these and many other examples, a segregation of the trophic niche has been shown to be an important diversification driver. Radiations are known in other groups of organisms, such as lichen-forming fungi. However, very few studies have investigated their adaptive nature, and none have focused on the trophic niche. In this study, we explore the role of the trophic niche in a putative radiation of endemic species from the Macaronesian Region, the Ramalina decipiens group. The photobiont diversity was studied by Illumina MiSeq sequencing of the ITS2 region of 197 specimens spanning the phylogenetic breadth and geographic range of the group. A total of 66 amplicon sequence variants belonging to the four main clades of the algal genus Trebouxia were found. Approximately half of the examined thalli showed algal coexistence, but in most of them, a single main photobiont amounted to more than 90% of the reads. However, there were no significant differences in photobiont identity and in the abundance of ITS2 reads across the species of the group. We conclude that a segregation of the trophic niche has not occurred in the R. decipiens radiation.

The fruticose genera in the Ramalinaceae (Ascomycota, Lecanoromycetes): their diversity and evolutionary history.

We present phylogenetic analyses of the fruticose Ramalinaceae based on extensive collections from many parts of the world, with a special focus on the Vizcaíno deserts in north-western Mexico and the coastal desert in Namibia. We generate a four-locus DNA sequence dataset for accessions of Ramalina and two additional loci for Niebla and Vermilacinia. Four genera are strongly supported: the subcosmopolitan Ramalina, the new genus Namibialina endemic to SW Africa, and a duo formed by Niebla and Vermilacinia, endemic to the New World except the sorediate V. zebrina that disjunctly occurs in Namibia. The latter three genera are restricted to coastal desert and chaparral where vegetation depends on moisture from ocean fog. Ramalina is subcosmopolitan and much more diverse in its ecology. We show that Ramalina and its sister genus Namibialina diverged from each other at c. 48 Myrs, whereas Vermilacinia and Niebla split at c. 30 Myrs. The phylogeny of the fruticose genera remains unresolved to their ancestral crustose genera. Species delimitation within Namibialina and Ramalina is rather straightforward. The phylogeny and taxonomy of Vermilacinia are fully resolved, except for the two youngest clades of corticolous taxa, and support current taxonomy, including four new taxa described here. Secondary metabolite variation in Niebla generally coincides with major clades which are comprised of species complexes with still unresolved phylogenetic relationships. A micro-endemism pattern of allopatric species is strongly suspected for both genera, except for the corticolous taxa within Vermilacinia. Both Niebla and saxicolous Vermilacinia have chemotypes unique to species clades that are largely endemic to the Vizcaíno deserts. The following new taxa are described: Namibialina gen. nov. with N. melanothrix (comb. nov.) as type species, a single new species of Ramalina (R. krogiae) and four new species of Vermilacinia (V. breviloba, V. lacunosa, V. pustulata and V. reticulata). The new combination V. granulans is introduced. Two epithets are re-introduced for European Ramalina species: R. crispans (= R. peruviana auct. eur.) and R. rosacea (= R. bourgeana auct. p.p). A lectotype is designated for Vermilacinia procera. A key to saxicolous species of Vermilacinia is presented.

Physiological response of the bioindicator Ramalina farinacea in relation to atmospheric deposition in an urban environment.

Urban air pollution is one of the most important environmental problems. Lichens are good bioindicators in air pollution studies because of their dependence on the atmospheric deposition for nutrition. The present study focused on the effects of urbanization on the composition of atmospheric deposition inputs and physiological parameters of transplanted epiphytic lichen Ramalina farinacea. The status of lichens responded to urban pollutants related to vehicle and industrial activity (NO3-, Ca2+, Mg2+, NO2, PM10). The content of chlorophyll and FV/FM were positively related to the amount of precipitation (mm) and the Normalized Difference Vegetation Index (NDVI). The increase in lipid peroxidation and electrolyte conductivity, indicating damage to the cell membrane, was found in lichens transplanted to the urban environment. The high variability in electrolyte conductivity indicated that cell membrane injuries were characteristics of the investigated study area.

Toxicological effects of Ramalina aspera (lichen) on Biomphalaria glabrata snails and Schistosoma mansoni cercariae.

In this study, the molluscicidal activities against Biomphalaria glabrata and cercaricidal activities against Schistosoma mansoni of the ether extract of Ramalina aspera were evaluated. Additionally, toxicity parameters were evaluated at sublethal doses in terms of the influence of the extract on the fertility and fecundity of snails, as well as morphological alterations and quantification of their immunological cells. A test with Artemia salina was also carried out, in order to verify the environmental toxicity of the compound. The ether extract of R. aspera, in which divaricatic acid was identified as the major compound, demonstrated molluscicidal activity at low concentrations against both embryos (LC90 of 22.78, 24.23, 16.63 and 16.03 µg mL-1 for the gastrula, blastula, trochophore and veliger, respectively) and against adult snails (LC90 of 8.66 µg mL-1), after 24 h of exposure. At the sublethal doses, it was possible to observe a decrease in fecundity and quantitative and morphological changes in the defense cells of the exposed snails. In addition, the extract of R. aspera showed a cercaricidal effect on S. mansoni from the concentration of 5.0 µg mL-1, while showing low toxicity to Artemia salina. The ether extract of R. aspera demonstrated effective molluscicidal activity on embryos and adult snails of the species B. glabrata, cercariae of S. mansoni, and presenting low toxicity on Artemia salina. In this way, it could be considered a promising compound in the development of future molluscicidal and cercaricidal agents, thus helping to combat schistosomiasis.

The conservation of polyol transporter proteins and their involvement in lichenized Ascomycota.

In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast' polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated proteins in each cluster. Consequently, the number of sequences similar to characterized yeast' polyol transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have expanded during the evolution of Lecanoromycetes.

Characterization of the responses to saline stress in the symbiotic green microalga Trebouxia sp. TR9.

MAIN CONCLUSION: For the first time we provide a study on the physiological, ultrastructural and molecular effects of salt stress on a terrestrial symbiotic green microalga, Trebouxia sp. TR9. Although tolerance to saline conditions has been thoroughly studied in plants and, to an extent, free-living microalgae, scientific data regarding salt stress on symbiotic lichen microalgae is scarce to non-existent. Since lichen phycobionts are capable of enduring harsh, restrictive and rapidly changing environments, it is interesting to study the metabolic machinery operating under these extreme conditions. We aim to determine the effects of prolonged exposure to high salt concentrations on the symbiotic phycobiont Trebouxia sp. TR9, isolated from the lichen Ramalina farinacea. Our results suggest that, when this alga is confronted with extreme saline conditions, the cellular structures are affected to an extent, with limited chlorophyll content loss and photosynthetic activity remaining after 72 h of exposure to 5 M NaCl. Furthermore, this organism displays a rather different molecular response compared to land plants and free-living halophile microalgae, with no noticeable increase in ABA levels and ABA-related gene expression until the external NaCl concentration is raised to 3 M NaCl. Despite this, the ABA transduction pathway seems functional, since the ABA-related genes tested are responsive to exogenous ABA. These observations could suggest that this symbiotic green alga may have developed alternative molecular pathways to cope with highly saline environments.

Laboratory assessment of divaricatic acid against Biomphalaria glabrata and Schistosoma mansoni cercariae.

In this study, the molluscicidal and antiparasitic activities of divaricatic acid was evaluated, targeting the mollusc Biomphalaria glabrata and cercariae of the helminth Schistosoma mansoni. In addition, the environmental toxicity of divaricatic acid was assessed by bioassay using the microcrustacean Artemia salina. Divaricatic acid showed high toxicity against both adult snails (5µg/mL) and embryos (20µg/mL after 6h of exposure). Similar activity was observed in Schistosoma mansoni cercariae after only a short exposure time (10µg/mL after 30min of exposure). The divaricatic acid did not show toxicity in the acute test using Artemia salina at concentrations equal to or below 200µg/mL. The divaricatic acid proved to be a promising substance for the elimination of the snail Biomphalaria glabrata, an intermediate host of schistosomiasis, as well as the cercariae of the pathogen, while being non-toxic to the Artemia salina at the same concentrations. This is the first experimental observation of the molluscicidal and cercaricide activity of divaricatic acid.

Ramalina capitata (Ach.) Nyl. acetone extract: HPLC analysis, genotoxicity, cholinesterase, antioxidant and antibacterial activity.

In the present investigation, effects of Ramalina capitata acetone extract on micronucleus distribution on human lymphocytes, on cholinesterase activity and antioxidant activity (by the CUPRAC method) were examined, for the first time as well as its HPLC profile. Additionally, total phenolic compounds (TPC), antioxidant properties (estimated via DPPH, ABTS and TRP assays) and antibacterial activity were determined. The predominant phenolic compounds in this extract were evernic, everninic and obtusatic acids. Acetone extract of R. capitata at concentration of 2 µg mL-1 decreased a frequency of micronuclei (MN) for 14.8 %. The extract reduces the concentration of DPPH and ABTS radicals for 21.2 and 36.1 % (respectively). Values for total reducing power (TRP) and cupric reducing capacity (CUPRAC) were 0.4624 ± 0.1064 µg ascorbic acid equivalents (AAE) per mg of dry extract, and 6.1176 ± 0.2964 µg Trolox equivalents (TE) per mg of dry extract, respectively. The total phenol content was 670.6376 ± 66.554 µg galic acid equivalents (GAE) per mg of dry extract. Tested extract at concentration of 2 mg mL-1 exhibited inhibition effect (5.2 %) on pooled human serum cholinesterase. The antimicrobial assay showed that acetone extract had inhibition effect towards Gram-positive strains. The results of manifested antioxidant activity, reducing the number of micronuclei in human lymphocytes, and antibacterial activity recommends R. capitata extract for further in vivo studies.

The role of native lichens in the biomonitoring of gaseous mercury at contaminated sites.

Contamination by atmospheric mercury has been assessed in two different areas from Spain (Las Cuevas, Ciudad Real and Flix, Tarragona) using lichens as biomonitors. The relationship established between mercury contents in the soils and the gaseous mercury (GM) was also observed. It was found that the GM is highest in the vicinity of the source and it is dispersed depending on of the distance to the source and the wind directions. The mercury concentration in the gas phase in Flix was higher than that found in Las Cuevas and also higher than the value that the US EPA recommended. The mercury bioaccumulation in the native lichens from genders Ramalina and Xanthoria were used as biomonitors for absorbing mercury in Las Cuevas and Flix, respectively. The mercury uptake by Ramalina was higher than the amount accumulated by Xanthoria, a difference that was mainly due to the lichen characteristics. The content of mercury in lichens in relation to the mercury in gas was fitted by a Freundlich type equation, indicating that the equilibrium between both phases was established. Besides, transplanted Ramalina lichen in Las Cuevas allowed to obtain the kinetic of mercury uptake. A kinetic model of first order based on the equilibrium was proposed and the mass transfer constants for each sampling station were estimated. As it was expected, these values increased with the predominant wind flow direction.

Constituents of Ramalina capitata (Ach.) Nyl. extracts.

The aim of this work was to determine the chemical composition of the ether, ethyl acetate and dichloromethane extracts of Ramalina capitata by GC-FID and GC-MS for the first time. The main identified components in the ether, ethyl acetate and dichloromethane extracts were everninic acid (24.7, 33.7 and 22.2%), orcinol (25.8, 16.7 and 11.9%), orcinol monomethyl ether (11.6, 7.6 and 4.8%), 3-methylorsellinic acid (10.2, 7.1 and 9.0%) and usnic acid (4.4, 8.2 and 25.8%), respectively. Considerable amount (10.4%) of 2-hydroxy-4-methoxy-3,5,6-trimethylbenzoic acid was detected in ethyl acetate extract. Additionally, palmitic acid, linoleic acid, oleic acid and stearic acid and their esters were observed in the dichloromethane and ethyl acetate extracts in the range of 4.1-0.1%, while their amount was below 0.05% in the ether extract. According to the results reported here R. capitata predominantly biosynthesises derivatives of orcinol and, in much lesser extent, derivatives of ß-orcinol.