HPLC purification of antioxidant and antibacterial peptides from a lichen "Parmotrema perlatum (Huds.) M. Choisy": Identification by LC-MS/MS peptide mass fingerprinting.

Parmotrema perlatum, a lichen belonging to the family Parmeliaceae, is well known for its culinary benefits and aroma used as a condiment in Indian homes is also known as the "black stone flower" or "kalpasi" in India. This research intends to analyze the antioxidant power of the crude extracts using four pH-based buffers solubilized proteins/peptides and RP-HPLC fractions of P. perlatum obtained by purification. The proteins that were extracted from the four different buffers were examined using LC-MS/MS-based peptide mass fingerprinting. When compared to the other buffers, the 0.1 M of Tris-HCl buffer pH 8.0 solubilized proteins/peptides had the strongest antioxidant capacity. The sequential purification of the peptide was carried out by using a 3-kDa cut-off membrane filter and semipreparative RP-HPLC. Additionally, the purified fractions of the peptide's antioxidant activity were assessed, and effects were compared with those of the crude and 3 kDa cut--off membrane filtrates. The peptide fractions were sequenced by LC-MS/MS, which reveals that fraction 2 from RP-HPLC with the sequence LSWFMVVAP has shown the highest antioxidant potential in comparison with other fractions which can serve as the potential natural antioxidant drug. Further, fraction 2 also showed antibacterial activity against the selected microorganisms.

Towards the Use of Lichens as a Source of Bioactive Substances for Topical Applications.

The increasing incidence of dermatological diseases prompts the search for new natural methods of treatments, and lichens, with their special symbiotic structure, are a little-known and promising source of biologically active substances. Seven lichen species, Cladonia unicialis (L.) Weber ex F.H. Wigg. (Cladoniaceae), Evernia prunastri (L.) Ach. (Parmeliaceae), Hypogymnia physodes (L.) Nyl. (Parmaliaceae), Parmelia sulcata (Taylor) (Parmeliaceae), Physcia adscendens (Fr.) H. Olivier (Physciaceae), Pseudoevernia furfuracea (L.) Zopf (Parmeliaceae), and Xanthoria parietina (L.) Th. Fr. (Teloschistaceae), were used in our experiment. We identified different metabolites in the acetone extracts of all the lichen species. Based on the high-performance liquid chromatography analysis, the content of lichen substances in the extracts was evaluated. The impact of the individual lichen-specific reference substances, compared to the lichen extracts, on the viability of keratinocytes (HaCaT cell line) and fibroblasts (BJ cell line) and on the activity of selected skin-related enzymes was investigated. Our results revealed that only emodin anthrone at a concentration of 200 mg/L was cytotoxic to keratinocytes and fibroblasts in both cell viability assays. In turn, the C. uncialis extract was only cytotoxic to keratinocytes when used at the same concentration. The other tested treatments showed a positive effect on cell viability and no cytotoxicity or indeterminate cytotoxicity (shown in only one of the tests). Elastase and collagenase activities were inhibited by most of the lichen extracts. In turn, the individual lichen compounds (with the exception of evernic acid) generally had an undesirable stimulatory effect on hyaluronidase and collagenase activity. In addition, almost all the tested compounds and extracts showed anti-inflammatory activity. This suggests that some lichen compounds hold promise as potential ingredients in dermatological and skincare products, but their safety and efficacy require further study. The high cytotoxicity of emodin anthrone highlights its potential use in the treatment of hyperproliferative skin diseases such as psoriasis.

Two-step isolation of the two major paraconic acids of Cetraria islandica.

The lichen Cetraria islandica is traditionally used as a demulcent for the symptomatic treatment of irritations of the mouth and throat and associated dry cough, as well as for the treatment of temporary loss of appetite. In addition to depsides and depsidones, thalli contain paraconic acids, a group of secondary metabolites commonly found in lichens and fungi. Among those, protolichesterinic acid has shown promising pharmacological activities. However, the efficient isolation of paraconic acids is quite complex due to their very similar chemical structures and their weak ultraviolet absorption. In the present work, a two-step isolation protocol of protolichesterinic acid and lichesterinic acid from a complex paraconic acid mixture is described using Sephadex LH20 column chromatography and fast centrifugal partition chromatography. Final purities higher than 95% and recoveries above 50% are achieved. Additionally, reliable qualitative techniques for detecting and differentiating paraconic acids are described. Finally, some data on compound stability and enantiomeric purity are shown.

Two New α-Glucosidase Inhibitory Depsidones from the Lichen Parmotrema cristiferum (Taylor) Hale.

A bioactivity-guided investigation of the lichen Parmotrema cristiferum (Taylor) Hale (Parmeliaceae) led to the isolation of two new depsidones, cristifones A and B (1 and 2). The structures of the isolated compounds were identified by spectroscopic methods and comparison with the literature data. Compound 1 showed the initial combined structures of depsidone and depside cores. The two isolated compounds were then evaluated for α-glucosidase inhibition. Compounds 1 and 2 were confirmed as potent, with IC50 values of 21.5 and 18.4 µM, respectively. Compound 2 was a non-competitive inhibitor against α-glucosidase, as indicated by the intersect in the second quadrant of each respective plot.

Ultrastructural, Energy-Dispersive X-ray Spectroscopy, Chemical Study and LC-DAD-QToF Chemical Characterization of Cetraria islandica (L.) Ach.

The lichen Cetraria islandica (L.) Ach. has been used in traditional and modern medicines for its many biological properties such as immunological, immunomodulating, antioxidant, antimicrobial, and anti-inflammatory activities. This species is gaining popularity in the market, with interest from many industries for selling as medicines, dietary supplements, and daily herbal drinks. This study profiled the morpho-anatomical features by light, fluorescence, and scanning electron microscopy; conducted an elemental analysis using energy-dispersive X-ray spectroscopy; and phytochemical analysis was performed using high-resolution mass spectrometry combined with a liquid chromatography system (LC-DAD-QToF) of C. islandica. In total, 37 compounds were identified and characterized based on comparisons with the literature data, retention times, and their mass fragmentation mechanism/s. The identified compounds were classified under five different classes, i.e., depsidones, depsides, dibenzofurans, aliphatic acids, and others that contain simple organic acids in majority. Two major compounds (fumaroprotocetraric acid and cetraric acid) were identified in the aqueous ethanolic and ethanolic extracts of C. islandica lichen. This detailed morpho-anatomical, EDS spectroscopy, and the developed LC-DAD-QToF approach for C. islandica will be important for correct species identification and can serve as a useful tool for taxonomical validation and chemical characterization. Additionally, chemical study of the extract of C. islandica led to isolation and structural elucidation of nine compounds, namely cetraric acid (1), 9'-(O-methyl)protocetraric acid (2), usnic acid (3), ergosterol peroxide (4), oleic acid (5), palmitic acid (6), stearic acid (7), sucrose (8), and arabinitol (9).

Paresordin A, a new diphenyl cyclic peroxide from the lichen Parmotrema praesorediosum.

From the lichen Parmotrema praesorediosum, one new diphenyl peroxide, named praesordin A (1), together with four depsidones, including virensic acid (2), protocetraric acid (3), 8'-O-methylprotocetraric acid (4), and furfuric acid (5) were purified. Their structures were chacracterized using extensive HR-ESI-MS and NMR spectroscopic methods. The isolated compounds (2-5) possessed stronger α-glucosidase inhibitory activity (IC50 = 43.7-110.1 µM) than the standard drug acarbose (IC50 = 214.5 µM).

The Genus Cetraria s. str.-A Review of Its Botany, Phytochemistry, Traditional Uses and Pharmacology.

The genus Cetraria s. str. (Parmeliaceae family, Cetrarioid clade) consists of 15 species of mostly erect brown or greenish yellow fruticose or subfoliose thallus. These Cetraria species have a cosmopolitan distribution, being primarily located in the Northern Hemisphere, in North America and in the Eurasia area. Phytochemical analysis has demonstrated the presence of dibenzofuran derivatives (usnic acid), depsidones (fumarprotocetraric and protocetraric acids) and fatty acids (lichesterinic and protolichesterinic acids). The species of Cetraria, and more particularly Cetraria islandica, has been widely employed in folk medicine for the treatment of digestive and respiratory diseases as decoctions, tinctures, aqueous extract, and infusions. Moreover, Cetraria islandica has had an important nutritional and cosmetic value. These traditional uses have been validated in in vitro and in vivo pharmacological studies. Additionally, new therapeutic activities are being investigated, such as antioxidant, immunomodulatory, cytotoxic, genotoxic and antigenotoxic. Among all Cetraria species, the most investigated by far has been Cetraria islandica, followed by Cetraria pinastri and Cetraria aculeata. The aim of the current review is to update all the knowledge about the genus Cetraria covering aspects that include taxonomy and phylogeny, morphology and distribution, ecological and environmental interest, phytochemistry, traditional uses and pharmacological properties.

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.

Assessment of air pollution at the indoor environment of a shooting range using lichens as biomonitors.

The aim of the study was to examine the changes in ecophysiological parameters and accumulation of heavy metals in lichens transplanted to a shooting-range environment. Thalli of the epiphytic lichen were transplanted from an unpolluted site to a shooting range. Chlorophyll a fluorescence, thiobarbituric acid reactive substances (TBARS), and damage to cell membranes in the lichen Evernia prunastri and Ramalina farinacea thalli were determined after 3 months exposure period indoors in a shooting range. The concentrations of some heavy metals including cadmium, copper, iron, manganese, nickel, lead, antimony, and zinc were measured in lichens as indicators of the levels of air pollution in the studied environment. Thalli of the lichens transplanted to the shooting-range environment showed stress symptoms where the presence of metal pollutants produced a loss of integrity of lichen cell membranes and induced oxidative stress as evidenced by increased levels of TBARS. The response of lichens transplanted to indoors of shooting range demonstrated a significant accumulation of Pb indicating potential increased metal exposure and consequent adverse health effects.

Usnic acid suppresses cervical cancer cell proliferation by inhibiting PD-L1 expression and enhancing T-lymphocyte tumor-killing activity.

The programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway is abnormally expressed in cervical cancer cells. Moreover, PD-1/PD-L1 blockade reduces the apoptosis and exhaustion of T cells and inhibits the development of malignant tumors. Usnic acid is a dibenzofuran compound originating from Usnea diffracta Vain and has anti-inflammatory, antifungal, and anticancer activities. However, the molecular mechanism of its antitumor effects has not been fully elucidated. In this work, we first observed that usnic acid decreased the expression of PD-L1 in HeLa cells and enhanced the cytotoxicity of co-cultured T cells toward tumor cells. Usnic acid inhibited PD-L1 protein synthesis by reducing STAT3 and RAS pathways cooperatively. It was subsequently shown that usnic acid induced MiT/TFE nuclear translocation through the suppression of mTOR signaling pathways, and promoted the biogenesis of lysosomes and the translocation of PD-L1 to the lysosomes for proteolysis. Furthermore, usnic acid inhibited cell proliferation, angiogenesis, migration, and invasion, respectively, by downregulating PD-L1, thereby inhibiting tumor growth. Taken together, our results show that usnic acid is an effective inhibitor of PD-L1 and our study provide novel insights into the mechanism of its anticancer targeted therapy.

The Spasmolytic, Bronchodilator, and Vasodilator Activities of Parmotrema perlatum Are Explained by Anti-Muscarinic and Calcium Antagonistic Mechanisms.

Parmotremaperlatum is traditionally used in different areas of Pakistan to treat gastrointestinal, respiratory, and vascular diseases. This study evaluates the underlying mechanisms for traditional uses of P. perlatum in diarrhea, asthma, and hypertension. In vitro pharmacological studies were conducted using isolated jejunum, trachea, and aortic preparations, while the cytotoxic study was conducted in mice. Crude extract of P. perlatum(Pp.Cr), comprising appreciable quantities of alkaloids and flavonoids, relaxed spontaneously contracting jejunum preparation, K+ (80 mM)-induced, and carbachol (1 µM)-induced jejunum contractions in a concentration-dependent manner similar to dicyclomine and dantrolene. Pp.Cr showed a rightward parallel shift of concentration-response curves (CRCs) of Cch after a non-parallel shift similarto dicyclomine and shifted CRCs of Ca+2 to rightward much likeverapamil and dantrolene, demonstrating the coexistence of antimuscarinic and Ca+2 antagonistic mechanism. Furthermore, Pp.Cr, dicyclomine, and dantrolene relaxed K+ (80 mM)-induced and Cch (1 µM)-induced tracheal contractions and shifted rightward CRCs of Cch similar to dicyclomine, signifying the dual blockade. Additionally, Pp.Cr also relaxed the K+ (80 mM)-induced and phenylephrine (1 µM)-induced aortic contraction, similarly to verapamil and dantrolene, suggesting Ca+2 channel antagonism. Here, we explored for the first time thespasmolytic and bronchodilator effects of Pp.Crand whether they maybe due to the dual blockade of Ca+2 channels and muscarinic receptors, while the vasodilator effect might be owing to Ca+2 antagonism. Our results provide the pharmacological evidence that P. perlatum could be a new potential therapeutic option to treat gastrointestinal, respiratory, and vascular diseases. Hence, there is a need for further research to explore bioactive constituent of P. perlatum as well as further investigation by suitable experimental models are required to further confirm the importance and usefulness of P. perlatum in diarrhea, asthma, and hypertension treatment.

Antimicrobial and antioxidant activity of Evernia prunastri extracts and their isolates.

Lichens are symbiotic organisms formed by a fungus and one or more photosynthetic partners which are usually alga or cyanobacterium. Their diverse and scarcely studied metabolites facilitate adaptability to extreme living conditions. We investigated Evernia prunastri (L.) Ach., a widely distributed lichen, for its antimicrobial and antioxidant potential. E. prunastri was sequentially extracted by hexane (Hex), dichloromethane (DCM) and acetonitrile (ACN) that were screened for their antioxidant and antimicrobial (against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans) activities. The Hex extract possessed the highest antioxidant capacity (87 mg ascorbic acid/g extract) corresponding to the highest content of phenols (73 mg gallic acid/g extract). The DCM and Hex extracts were both active against S. aureus (MICs of 4 and 21 µg/ml, respectively) but were less active against Gram-negative bacteria and yeast. The ACN extract exhibited activity on both S. aureus (MIC 14 µg/ml) and C. albicans (MIC 38 µg/ml) and was therefore further fractionated by silica gel column chromatography. The active compound of the most potent fraction was subsequently characterized by 1H and 13C-NMR spectroscopy and identified as evernic acid. Structural similarity analyses were performed between compounds from E. prunastri and known antibiotics from different classes. The structural similarity was not present. Antioxidant and antimicrobial activities of E. prunastri extracts originate from multiple chemical compounds; besides usnic acid, most notably evernic acid and derivatives thereof. Evernic acid and its derivatives represent possible candidates for a new class of antibiotics.

HPLC Fingerprint Analysis with the Antioxidant and Cytotoxic Activities of Selected Lichens Combined with the Chemometric Calculations.

The aim of this study was to evaluate the ability of multivariate techniques to predict antioxidant and cytotoxic activity of the selected lichens from the chromatographic data. A simple and reproducible HPLC-DAD technique has been used to obtain the chromatographic fingerprint profiles. Reversed phase high performance liquid chromatography (RP-HPLC) linear gradient system with methanol, water and phosphoric acid (V) (pH 2.3) as the mobile phase was used (50 min). Principal Component Analysis (PCA) has been applied to the evaluation of the phytochemical similarity between studied samples, especially between the same species collected in various places of Poland (Cetraria islandica (L.) Ach., CI, Cladina mitis Sandst., CM, Hypogymnia physodes (L.) Nyl., HP). The ability to scavenge free radicals was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods and the total phenolic content was determined by Folin-Ciocalteu (F-C) test. In the case of DPPH % of inhibition was higher for selected species (Pseudevernia furfuracea (L.) Zopf, H. physodes in comparison to the literature data. The FRAP test showed that the H. physodes extract had higher ability to scavenge free radical in comparison to Cladonia furcata (Huds.) Schrader and Evernia prunastri (L.) Ach., whereas P. furfuracea extract showed higher ability than C. islandica. The high content of phenolics in P. furfuracea and H. physodes confirms their high antioxidant activity. The cytotoxic activity of studied extracts was tested by cell culture method using the human HL-60 / MX2 acute CKL-22 (CRL-2257) promyelocytic leukemia tumor cell line. The lowest values of IC50 [µg∙mL-1] were obtained for: H. physodes (HP1)-99.4; C. digitate-122.6; H. physodes (HP)-136.5, C. subulata-142.6; C. mitis-180.2.

Antibacterial activity of usnic acid from Usnea longissima Ach.

Isolation and identification of secondary metabolite from n-hexane fraction of lichen Usnea longissima Ach. and its bioactivity as antibacterial have been carried out. The isolation of chemical compounds was performed by using Gravity Column Chromatography (GCC), solvent systems as mobile phase (n-hexane, ethyl acetate), and Thin Layer Chromatography (TLC). The form of isolated compound was yellow needle crystals. The result of 1D-NMR (1H and 13C-NMR) data showed that the isolated compound was usnic acid. Antibacterial bioactivity testing was conducted by using the paper disk diffusion method indicated that the usnic acid compound was actively inhibited the growth of E. coli (ATCC35218) and S. aureus (ATCC25923) at the concentrations of 500mg/mL and 1000mg/mL with inhibition zone between 12mm and 17mm. In addition, S. typhi (YCTC) was only inhibited at the concentration of 1000mg/mL with inhibition zone of 14mm.

Lichens of Parmelioid Clade as Promising Multitarget Neuroprotective Agents.

Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease are multifactorial disorders which are increasing in incidence and prevalence over the world without existing effective therapies. The search for new multitarget compounds is the latter therapeutic strategy to address these pathological conditions. Lichens have an important and unknown therapeutic value attributed to their unique secondary metabolites. The aim of this study is to evaluate for the first time the in vitro neuroprotective activities and molecular mechanisms underlying methanol extracts of lichens of the parmelioid clade and to characterize major bioactive secondary metabolites responsible for their pharmacological actions. Of the 15 parmelioid lichen species, our results showed that Parmotrema perlatum and Hypotrachyna formosana methanol extracts exhibited high antioxidant activity as evidenced in ORAC, DPPH, and FRAP assays. Then, SH-SY5Y cells were pretreated with methanol extracts (24 h) followed by Fenton reagent exposure (2 h). Pretreatments with these two more antioxidant methanol lichen extracts increased cell viability, reduced intracellular ROS, prevented oxidative stress biomarkers accumulation, and upregulated antioxidant enzyme (CAT, SOD, GR, and GPx) activity compared to Fenton reagent cells. The neuroprotective activity was much higher for H. formosana than for P. perlatum, even equal to or higher than Trolox (reference compound). Moreover, H. formosana extracts inhibited both AChE and BuChE activities in a concentration dependent manner, and P. perlatum only showed concentration dependent activity against AChE. Finally, chemical composition analysis using TLC and HPLC methods revealed that physodic acid, lividic acid, and lichexanthone are major secondary metabolites in H. formosana and stictic acid and constictic acid are in P. perlatum. These results demonstrated that P. perlatum and, specially, H. formosana are promising multitargeted neuroprotective agents due to their antioxidant and AChE and BuChE inhibition activities.

Active Metabolites from Endolichenic Fungus Talaromyces sp.

The active metabolites investigation of Talaromyces sp. (strain No. MH551540) associated with Xanthoparmelia angustiphylla afforded one new δ-lactone, talaromycin A (1), together with six known compounds, clearanol A (2), 6-methylbiphenyl-3,3',4,5'-tetraol (3), desmethylaltenusin (4), ergone (5), ergosterol (6), and palmitic acid (7). The structures of these compounds were elucidated by a combination of spectroscopic-data interpretation and single-crystal X-ray diffraction analysis. The cytotoxicities of 1-7 and the antioxidant activities of 3 and 4 were also evaluated.

Effects of two lichen acids isolated from Pseudevernia furfuracea (L.) Zopf in cultured human lymphocytes.

The present study aims at assessing the efficacies of olivetoric acid (OA) and physodic acid (PA) isolated from Pseudevernia furfuracea (L.) Zopf (Parmeliaceae) in human lymphocytes (HLs) in vitro. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were performed to establish cytotoxicity in HLs. Besides, oxidative stress and genotoxicity were monitored by estimating the changes of total oxidative stress (TOS) and 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels, respectively, in HLs. At the same time, OA- and PA-induced total antioxidant capacity (TAC) levels in HLs were determined. Although especially low concentrations of OA (IC50=109.94 mg/L) and PA (IC50=665.49 mg/L) did not show cytotoxic effect at high levels in HLs, it was revealed that cytotoxicity was significantly (p<0.05) associated with oxidative stress and genotoxicity via correlation analysis. While TOS level in HLs did not statistically (p>0.05) increase in the presence of all treatments (0.5-100 mg/L) of PA, TAC level was increased by PA applications in certain concentrations (0.5-10 mg/L). Overall, the obtained data indicate that OA and especially PA as lichen compounds that do not cause oxidative stress can be a new resource of therapeutics as recognized in the present study with their high antioxidant features.

Morphological, Secondary Metabolite and ITS (rDNA) Variability within Usnic Acid-Containing Lichen Thalli of Xanthoparmelia Explored at the Local Scale of Rock Outcrop in W-Alps.

Lichen secondary metabolites (LSMs) are regarded with interest for valuable biological properties, but chemical variability among/within lichen taxa has been only fragmentarily characterized by advanced analytical techniques. Knowledge of variability at a local geographic scale has been particularly neglected, while it should address the collection of chemically homogeneous materials to test and exploit LSMs. Here we evaluated the chemical variability of 48 Xanthoparmelia specimens from two rock outcrops in Western Italian Alps, representative of nine morphotypes and sixteen rDNA ITS haplotypes. Qualitative and quantitative analyses were performed by HPLC-DAD-ESI-MS2 and UPLC-HDR-DAD, respectively, and revealed the occurrence of 18 LSMs. Chemical partition allowed distinguishing six chemical groups, only partially overlapping with distinct morphotypes and three divergent haplotype groups, which, overall, accounted for the co-occurrence of different taxa only in part identifiable with species described for Europe. Some morphotypes were variable in presence and concentration of LSMs, and chemical divergences also characterized single ITS haplotypes. Accordingly, the collection of chemically homogeneous materials, even at a local scale, may be not properly addressed by morphological features and ITS barcoding, and should be confirmed by a specimen-level chemical characterization.

Metabolomic Analysis of Two Parmotrema Lichens: P. robustum (Degel.) Hale and P. andinum (Mull. Arg.) Hale Using UHPLC-ESI-OT-MS-MS.

Lichens are symbiotic associations of fungi with microalgae and/or cyanobacteria. Lichens belonging to the Parmeliaceae family comprise 2700 species of lichens, including the Parmotrema genus which is composed of 300 species. The metabolites of this genus include depsides, depsidones, phenolics, polysaccharides, lipids, diphenylethers and dibenzofurans, which are responsible for the biological activities reported including antidiabetic, antihelmintic, anticancer, antioxidant, antibacterial, anti-inflammatory, antimitotic, antitumoral, antifungal, and antioxidant enzyme inhibitory. Due to scarce knowledge of metabolomic profiles of Parmotrema species (P. andinum and P. robustum), a full metabolome study based on ultra-high performance liquid chromatography- diode array detector-electrospray ionization-quadrupole-orbitrap-mass-spectrometry (UHPLC-DAD-ESI-Q-orbitrap MS) was performed for a comprehensive characterization of their substances. From the methanolic extracts of these species, a total of 54 metabolites were identified for the first time using this hyphenated technique, including thirty compounds in P. andinum, and thirty-seven in P. robustum. Moreover, two compounds were not identified as known compounds, and could be new structures, according to our data. This report shows that this technique is effective and accurate for rapid chemical identification of lichen substances and the compounds identified could serve as chemotaxonomic markers to differentiate these ruffle lichens.

Protolichesterinic Acid, Isolated from the Lichen Cetraria islandica, Reduces LRRC8A Expression and Volume-Sensitive Release of Organic Osmolytes in Human Lung Epithelial Cancer Cells.

We have tested the effect of protolichesterinic acid (PA) on the activity of the volume-sensitive release pathway for the organic osmolyte taurine (VSOAC) and the expression of the leucine-rich-repeat-channel 8A (LRRC8A) protein, which constitutes an essential VSOAC component. Exposing human lung cancer cells (A549) to PA (20 µg/mL, 24 h) reduces LRRC8A protein expression by 25% and taurine release following osmotic cell swelling (320 → 200 mOsm) by 60%. C75 (20 µg/mL, 24 h), a γ-lactone with a C8 carbon fatty acid chain, reduces VSOAC activity by 30%, i.e. less than PA. Stearic acid (20 µg/mL, 24 h) has no effect on VSOAC. Hence, length of PA's fatty acid chain adds to γ-lactone's inhibitory action. 5-Lipoxygenase (5-LO) activity is essential for swelling-induced activation of VSOAC. PA has no effect on cellular concentration of leukotrienes (5-HETE/LTB4 ) under hypotonic conditions, excluding that PA mediated inhibition of VSOAC involves 5-LO inhibition. A549 cells exposed to the chemotherapeutic drug cisplatin (10 µM, 24 h) reveal signs of apoptosis, i.e. 25% reduction in cell viability as well as 1.3-, 1.5- and 3.3-fold increase in the expression of LRRC8A, Bax (regulator of apoptosis) and p21 (regulator of cell cycle progression), respectively. PA reduces cell viability by 30% but has no effect on p21/Bax expression. This excludes PA as a pro-apoptotic drug in A549 cells.