Liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry characterization of depsides and depsidones from the Chilean lichen Parmotrema perlatum.

Lichens are recognized by their unique compounds and diverse applications in food, medicines, and cosmetics. Using ultra-high pressure liquid chromatography, coupled with a high-resolution mass spectrometer, metabolomic profiling of the lichen Parmotrema perlatum, from a methanolic extract, was performed. Based on characteristic fragmentation patterns, twenty-five lichenic substances were tentatively identified including 5 depsides, 12 depsidones, 2 diphenyl ethers, 1 aromatic considered as possible artifact, 1 dibenzofuran, 1 carbohydrate, 1 organic acid, and 2 undefined compounds. To the best of our knowledge, this is a more complete report of their phytochemistry from P perlatum. Our findings of the P perlatum profile may contribute and complement the current data of the Parmotrema genus.

Contribution of Mine-Derived Airborne Particulate Matter to Ca, Fe, Mn and S Content and Distribution in the Lichen Punctelia hypoleucites Transplanted to Bajo de la Alumbrera Mine, Catamarca (Argentina).

The aim of this work was to relate the contribution of mine-derived airborne particulate matter to Ca, Fe, Mn and S content and distribution in Punctelia hypoleucites transplanted to Bajo de la Alumbrera, an important open-pit mine in Catamarca, Argentina. Lichen samples were transplanted to four monitoring sites: two sites inside the mine perimeter and two sites outside the mine. After three months, elemental distribution in samples was analyzed by microparticle-induced X-ray emission (microPIXE), and elemental concentration was determined by specific techniques: Ca and Fe by instrumental neutron activation analysis, Mn by inductively coupled plasma atomic emission spectrometry and S by a turbidimetric method. A differential distribution of S and Ca in thalli transplanted in-mine sites was detected compared to that of samples transplanted outside-mine sites. An overlap of Fe and S in the upper cortex of the apothecium section was observed, leading to infer a mineral association of both elements. Similar association was observed for Ca and S. In addition to these results, the significantly higher concentration detected for S and Mn in in-mine site samples suggests a contribution of Fe, S, Ca and Mn of mining origin to the content and distribution of these elements in P. hypoleucites. MicroPIXE complemented with Mössbauer spectroscopy analysis determined the presence of pyrite particles together with other iron-bearing minerals displaying different degrees of oxidation. These results point to a mining origin of the airborne particulate matter trapped by the lichen thalli transplanted to Bajo de la Alumbrera. These findings indicate that P. Hypoleucites acts as an excellent air quality biomonitor in the Bajo de la Alumbrera mine area.

Dibenzofurans from nature: Biosynthesis, structural diversity, sources, and bioactivities.

Dibenzofurans are a small class of natural products with versatile biological activities that used to be thought to come mainly from lichens and ascomycetes. In fact, they are also distributed widely in higher plants, especially in the families Rosaceae and Myrtaceae. Dibenzofurans and derivatives from lichens and ascomycetes have been well reviewed, but dibenzofurans from all biological sources in nature have not been reviewed. In this review, dibenzofurans from all natural sources have been comprehensively reviewed, and a total of 211 dibenzofurans isolated and identified from organisms between 1843 and March 2023 are categorized and discussed, including their biosynthesis, structural diversity, sources, and bioactivities.

Parietin, the Vibrant Natural Dye in Xanthoria parietina.

The use of natural dyes in several areas is regulated by current European and non-European legislation, due to various problems with synthetic dyes. The analysis revealed that the lichen studied: Xanthoria parietina has potential natural dye sources and provides bright colors for extraction solvents. Furthermore, dyed wool and toile fabric have good fastness properties in ammonia fermentation and boiling water, both with and without mordants. The sample dyes with Xanthoria parietina were characterized by several analytical techniques: high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization with tandem mass spectrometry (HPLC-ESI-Q-ToF). As compounds from Xanthoria parietina form a complex with mordants and tissues, it is impossible to identify the molecules responsible for coloring using chromatographic techniques. However, we have evaluated the dyeing power of their major molecule, parietin. To further confirm the coloring power of the isolated parietin molecule, we performed a dye test with pure parietin. Thus, CIALAB analyses have shown parietin is the molecule responsible for the coloring obtained by Xanthoria parietina. The utilization of parietin derived from lichens facilitates the development of sustainable dyes for textile coloring, presenting an environmentally friendly alternative to synthetic dyes while simultaneously enriching lichen biodiversity.

Evernia prunastri lichen as a source of bioactive glucans with potential for topical applications.

Microwave hydrothermal treatment was selected to extract valuable fractions with bioactive and gelling properties from Evernia prunastri lichen with potential for topical applications. The impact of the extraction processing conditions on the soluble extracts, mucilage fraction and residual solid phase was analyzed within a lichen global valorization approach. A particular stress was made on the thermo-rheological and structural characteristics of the extracted glucan and galactomannan polymers, the corresponding gelled matrices, and their cosmetic feasibility. Results revealed that the proposed microwave-assisted treatment showed a relevant influence on the phytochemical features of the aqueous soluble extracts, accounting the major protein content at 120 °C and the enhanced antioxidant and anti-tyrosinase properties at 140 °C. Extracts at 200 °C showed the highest anti-inflammatory (COX-1 and COX-2 inhibition) efficacies. The biopolymer analyses indicated that those recovered after lichen hydrothermal treatment at 160 °C featured a good extraction performance, the highest molecular weight, apparent viscosity, and antiproliferative potential. The thermo-rheological properties of the corresponding matrices formulated at 10 % and 60 or 80 °C exhibited the strongest and most thermo-reversible characteristics, as well as antifreezing feasibility. Another advantage of the selected fractions was the absence of skin irritation according to the in vitro skin irritation assay.

Usnea aurantiaco-atra (Jacq) Bory: Metabolites and Biological Activities.

BACKGROUND: Lichens are complex symbiotic associations between a fungus and an alga or cyanobacterium. Due to their great adaptability to the environment, they have managed to colonize many terrestrial habitats, presenting a worldwide distribution from the poles to the tropical regions and from the plains to the highest mountains. In the flora of the Antarctic region, lichens stand out due to their variety and development and are a potential source of new bioactive compounds. METHODS: A phytochemical study of the Antarctic lichen Usnea aurantiaco-atra (Jacq) Bory was conducted with the intention of determining the most important metabolites. In addition, the cytotoxic and antioxidant activities of its extracts were determined. RESULTS: Cytotoxicity studies revealed that the hexane extract contains usnic acid as a majority metabolite, in addition to linoleic acid, ergosterols and terpenes, and demonstrates cytotoxic activity against an A375 melanoma cell line. On the other hand, the presence of total phenols in the extracts did not influence their antioxidant activity. CONCLUSIONS: U. aurantiaco-atra contains mainly usnic acid, although there are terpenes and ergosta compounds that could be responsible for its cytotoxic activity. The presence of phenols did not confer antioxidant properties.

In†Vitro and in†Silico Studies of Lichen Compounds Atranorin and Salazinic Acid as Potential Antioxidant, Antibacterial and Anticancer Agents.

Lichens are symbiotic organisms made up of alga/cyanobacterium and fungus. We investigated antioxidant, antibacterial and anticancer properties of two lichen compounds, atranorin and salazinic acid, and five lichen species: Heterodermia boryi, Heterodermia diademata, Heterodermia hypocaesia, Parmotrema reticulatum, and Stereocaulon foliolosum. Free radical scavenging, Ferric reducing potential, Nitric oxide scavenging, and Trolox equivalent capacity were used to measure antioxidant activity. Strong radical scavenging action was demonstrated by atranorin and salazinic acid, with IC50 values of 39.31 µM and 12.14 µM, respectively. The Minimum Inhibitory Concentration (MIC) assay based on resazurin, was used to measure antibacterial activity. Parmotrema reticulatum demonstrated significant antibacterial activity against Raoultella planticola with MIC of 7.8 µg/mL. Cytotoxicity assay on breast cancer cell line was used to assess anticancer activity. To further understand the binding locations on the target proteins Er (Estrogen Receptor alpha), EGFR (Epidermal Growth Factor Receptor), mTOR (Mammalian Target of Rapamycin), and PgR (Progesterone Receptor), molecular docking experiments were conducted. Docking study showed that the binding energies of atranorin and salazinic acid with mTOR were -5.31 kcal/mol and -3.43 kcal/mol, respectively. The results suggest that atranorin has the potential to be a multitargeted molecule with natural antioxidant, antibacterial, and anticancer properties.

Dimeric (Poly)Hydroxynaphthazarins, Metabolites of Echinoderms and Lichens: The History of the Synthesis and Structure Elucidation.

This review provides information on the synthesis and revision of the structures of natural dimeric (poly)hydroxynaphthazarins, metabolites of echinoderms and lichens, and on the refinement of the direction and mechanism of reactions in the synthesis of some of these compounds.

Isolation of Mycosporine-like Amino Acids from Red Macroalgae and a Marine Lichen by High-Performance Countercurrent Chromatography: A Strategy to Obtain Biological UV-Filters.

Marine organisms have gained considerable biotechnological interest in recent years due to their wide variety of bioactive compounds with potential applications. Mycosporine-like amino acids (MAAs) are UV-absorbing secondary metabolites with antioxidant and photoprotective capacity, mainly found in organisms living under stress conditions (e.g., cyanobacteria, red algae, or lichens). In this work, five MAAs were isolated from two red macroalgae (Pyropia columbina and Gelidium corneum) and one marine lichen (Lichina pygmaea) by high-performance countercurrent chromatography (HPCCC). The selected biphasic solvent system consisted of ethanol, acetonitrile, saturated ammonium sulphate solution, and water (1:1:0.5:1; v:v:v:v). The HPCCC process for P. columbina and G. corneum consisted of eight separation cycles (1 g and 200 mg of extract per cycle, respectively), whereas three cycles were performed for of L. pygmaea (1.2 g extract per cycle). The separation process resulted in fractions enriched with palythine (2.3 mg), asterina-330 (3.3 mg), shinorine (14.8 mg), porphyra-334 (203.5 mg) and mycosporine-serinol (46.6 mg), which were subsequently desalted by using precipitation with methanol and permeation on a Sephadex G-10 column. Target molecules were identified by HPLC, MS, and NMR.

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).

137Cs and isotopic ratios of Pu and U in lichens and mosses from Russian Arctic areas.

Knowledge of past anthropogenic sources of radionuclide contamination in Russian Arctic areas is important to assess the radioecological situation of these less-studied regions. Therefore, we investigated the sources of radionuclide contamination in Russian Arctic in the 1990s. Lichen and moss samples were collected from 1993 to 1996 in Kola Peninsula, Franz Josef Land, and few other locations. The activity concentration of 137Cs was determined from the archived samples by gamma spectrometry in 2020. After radiochemical separation of Pu and U isotopes from the lichens and mosses, mass ratios 240Pu/239Pu, 234U/238U, 235U/238U, and 236U/238U were determined by mass spectrometry. 137Cs activity concentrations at the sampling date were found to vary from 3.1 ± 1.4 (Inari, Finnish-Russian border) to 303 ± 7 (Kola Peninsula) Bq/kg. The ranges of isotopic ratios were 0.0592 ± 0.0007 to 0.253 ± 0.082 for 240Pu/239Pu, (4.89 ± 3.91) × 10-5 to (6.86 ± 0.04) × 10-5 for 234U/238U, 0.0072104(21) to 0.007376(41) for 235U/238U, and from below 1 × 10-7 to (2.65 ± 0.19) × 10-6 for 236U/238U, respectively. Based on the measured isotopic ratios and characteristic isotopic ratios of known contamination sources, the main Pu and U sources in the sampled lichens and mosses are global fallout, the Chernobyl accident, and possibly local nuclear activities. These results contribute to further understanding of past nuclear events and resulting nuclear contamination in Russian Arctic terrestrial areas.

Lichen-Derived Actinomycetota: Novel Taxa and Bioactive Metabolites.

Actinomycetes are essential sources of numerous bioactive secondary metabolites with diverse chemical and bioactive properties. Lichen ecosystems have piqued the interest of the research community due to their distinct characteristics. Lichen is a symbiont of fungi and algae or cyanobacteria. This review focuses on the novel taxa and diverse bioactive secondary metabolites identified between 1995 and 2022 from cultivable actinomycetota associated with lichens. A total of 25 novel actinomycetota species were reported following studies of lichens. The chemical structures and biological activities of 114 compounds derived from the lichen-associated actinomycetota are also summarized. These secondary metabolites were classified into aromatic amides and amines, diketopiperazines, furanones, indole, isoflavonoids, linear esters and macrolides, peptides, phenolic derivatives, pyridine derivatives, pyrrole derivatives, quinones, and sterols. Their biological activities included anti-inflammatory, antimicrobial, anticancer, cytotoxic, and enzyme-inhibitory actions. In addition, the biosynthetic pathways of several potent bioactive compounds are summarized. Thus, lichen actinomycetes demonstrate exceptional abilities in the discovery of new drug candidates.

Elemental profile identifies metallurgical pollution in epiphytic lichen Xanthoria parietina and (hypo)xanthine correlates with metals.

The accumulation of 55 elements in lichens under the heap of a former nickel smelter (village Dolná Streda, Slovakia) and at eight sites at different distances from the heap plus six sites throughout Slovakia was studied to determine the elemental profile. The major metals in the heap sludge and in the lichens below the heap (Ni, Cr, Fe, Mn, and Co) were surprisingly low in lichens from both the near and far vicinity of the heap (4-25 km), indicating limited airborne spread. However, two different sites with metallurgical activity (another site near the ferroalloy producer in Orava) typically contained the highest amount of individual elements, including rare earth elements, Th, U, Ag, Pd, Bi and Be, and their separation from other sites was confirmed by PCA and HCA analyses. In addition, the amounts of Cd, Ba and Re were highest at sites without a clear source of pollution and further monitoring is needed. It was also an unexpected finding that the enrichment factor calculated using UCC values was increased (often considerably >10) for 12 elements at all 15 sites, indicating eventual anthropogenic contamination with P, Zn, B, As, Sb, Cd, Ag, Bi, Pd, Pt, Te and Re (and other EF values were locally increased). Metabolic analyses showed a negative correlation between some metals and metabolites (ascorbic acid, thiols, phenols and allantoin), but slightly positive (amino acids) or highly positive correlation with purine derivatives hypoxanthine and xanthine. The data suggest that lichens adapt their metabolism to excessive metal loading and that epiphytic lichens are suitable for identifying metal contamination even at apparently clean sites.

Lichen biomonitoring of seasonal outdoor air quality at schools in an industrial city in Thailand.

Poor air quality in school environments causes adverse health effects in children and decreases their academic performance. The main objective of this study was to use lichens as a biomonitoring tool for assessing outdoor air quality at schools in the industrial area of Laem Chabang municipality in Thailand. Thalli of the lichen Parmotrema tinctorum were transplanted from an unpolluted area to nine schools in the industrial area and to a control site. The lichens were exposed for four periods in the dry, hot, early rainy, and late rainy seasons, for 90 days each. The concentrations of 14 elements, including As, Cd, Co, Cr, Cu, Fe, Mn, Mo, Ni, Pb, Sb, Ti, V, and Zn, were determined using inductively coupled plasma‒mass spectrometry (ICP‒MS), and 8 physiological parameters were measured. The concentrations of all 14 investigated elements were clearly higher at the schools than at the control site. The contamination factors (CFs) suggested that 9 out of the 14 elements, including As, Cd, Co, Cr, Cu, Mo, Pb, Sb, and Ti, heavily contaminated the school environments, especially Pb, the concentration of which was 3 to 11 times higher than at the control site. The most polluted time was the hot season as evidenced by the investigated elements, and the least polluted time was the late rainy season. The pollution load indices (PLIs) demonstrated that schools in the inner and middle zones clearly had higher pollution loads than the schools in the outer zone during the rainy seasons, while the hot and dry seasons showed similar pollution levels in all zones. The vitality indices (VIs) showed that the lower lichen vitalities at most schools were observed during the dry season and at the schools in the inner and middle zones. Accordingly, the air performance indices (APIs) revealed that poorer air quality at most schools was found during the dry season and at the schools in the inner and middle zones. This study clearly showed that the transplanted lichen P. tinctorum was an effective bioindicator of air quality in school environments. The results illustrated that all studied schools were contaminated by air pollutants; therefore, improving air quality at the schools is crucial and should be an urgent issue for maintaining good health and may benefit children's academic achievements and careers in the long run.

Assessment of Cytotoxic/Antitumour Potential and in silico Study of Salazinic Acid Isolated from Parmotrema concurrens.

INTRODUCTION: Despite numerous scientific advances, cancer continues to be one of the main causes of death in the world. This situation has driven the search for promising molecules. Lichen substances have been widely described for their pharmacological potential. OBJECTIVE: The present study evaluated the antitumour potential of a depsidone isolated from Parmotrema concurrens- salazinic acid (SAL) - through in vitro, in vivo and in silico studies. METHODS: The molecule was isolated from the acetonic extract of the lichen and recrystallized in acetone. The macrophage J774, sarcoma-180 and MDA-MB-231 cell lines were used for the MTT cytotoxicity assay. The antitumor assay used a murine model (Swiss albino mice) with sarcoma-180. The animals were treated for seven consecutive days with doses of SAL (25 and 50 mg/kg) and 5-fluorouracil (20 mg/kg). RESULTS: Its purity was determined using high-performance liquid chromatography (94%), and its structure was confirmed by H1 and C13 nuclear magnetic resonance. SAL was not considered toxic to cancer cell lines, showing cell viability rates of 79.49 ± 4.15% and 86.88 ± 1.02% for sarcoma-180 and MDA-MB-231, respectively. The tumour inhibition rate was greater than 80% in the animals treated with SAL and 65% for those that received 5-fluorouracil. Simulations of molecular dynamics to estimate the flexibility of the interactions between human thymidylate synthase and derivatives of SAL and 5-fluorouracil revealed that SAL exhibited greater enzymatic interaction capacity, with highly favourable energy, compared to 5-fluorouracil. CONCLUSION: The present results demonstrate the potential of salazinic acid as a tumour inhibition agent.

NH3 concentrations below the current critical level affect the epiphytic macrolichen communities - Evidence from a Northern European City.

Acidophytic, oligotrophic lichens on tree trunks are widely considered to be the most sensitive biota to elevated concentrations of atmospheric ammonia (NH3). We studied the relationships between measured NH3 concentrations and the composition of macrolichen communities on the acidic bark of Pinus sylvestris and Quercus robur and the base-rich bark of Acer platanoides and Ulmus glabra at ten roadside and ten non-roadside sites in Helsinki, Finland. NH3 and nitrogen dioxide (NO2) concentrations were higher at the roadside than non-roadside sites indicating traffic as the main source of NH3 and nitrogen oxides (NOx). The diversity of oligotrophs on Quercus was lower at the roadside than non-roadside sites, while that of eutrophs was higher. The abundance and presence of oligotrophic acidophytes (e.g., Hypogymnia physodes) decreased with increasing NH3 concentration (2-year means = 0.15-1.03 µg m-3) especially on Q. robur, while those of eutrophic/nitrophilous species (e.g., Melanohalea exasperatula, Physcia tenella) increased. The abundance of some nitrophytes seemed to depend only on bark pH, i.e., their abundances were highest on Ulmus, which had the highest average bark pH. Overall, the results of lichen bioindicator studies may depend on tree species (bark pH) and lichen species used in calculating indices describing the air quality impact. Nevertheless, Quercus is recommended to be used to study the impact of NH3 alone and in combination with NOx on lichen communities, because the responses of both oligotrophic acidophytes and eutrophic species can already be observed at NH3 concentrations below the current critical level.

Synthesis, Characterization, and Bioactivity of the Lichen Pigments Pulvinamide, Rhizocarpic Acid, and Epanorin and Congeners.

The lichen natural products pulvinamide, rhizocarpic acid, and epanorin have been synthesized and characterized spectroscopically and by X-ray crystallography. The syntheses, by ring-opening of pulvinic acid dilactone (PAD), may well be biomimetic, given the well-known occurrence of PAD in lichen. The enantiomers, ent-rhizocarpic acid and ent-epanorin, and corresponding carboxylic acids, norrhizocarpic acid and norepanorin, were similarly prepared. All compounds were assessed for growth inhibitory activity against selected bacteria, fungi, a protist, a mammalian tumor cell line, and normal cells. Rhizocarpic acid is weakly antibacterial (Bacillus subtilis MIC = 50 µg/mL) and possesses modest but selective antitumor activity (NS-1 murine myeloma MIC = 3.1 µg/mL) with >10-fold potency relative to its enantiomer (MIC = 50 µg/mL).

Molecular docking and dynamics supported investigation of antiviral activity of Lichen metabolites of Roccella montagnei: an in silico and in vitro study.

Lichens are symbiotic organisms that have been traditionally used for treating different kinds of ailments. As there are only a few reports on the antiviral activity of lichens, we thought of evaluating the anti-Herpes simplex virus-1 (HSV-1) activity of methanolic extract of Roccella montagnei and their isolated compounds. Fractionation of crude methanolic extract of Roccella montagnei by column chromatography isolated two pure compounds. Antiviral activity was assessed using a CPE inhibition assay at non-cytotoxic concentrations on Vero cells. Molecular docking and dynamics studies were carried out against Herpes simplex type-1 thymidine kinase to understand the binding interactions of the isolated compounds with reference to acyclovir. Isolated compounds were characterized as methyl orsellinate and montagnetol by spectral methods. Methanolic extract of Roccella montagnei exhibited an EC50 value of 56.51 µg/ml, while the compounds methyl orsellinate and montagnetol offered EC50 values of 13.50 µg/ml and 37.52 µg/ml, respectively, against HSV-1 viral infection on Vero cell lines. The selectively index (SI) of montagnetol (10.93) was found to be higher when compared to that of methyl orsellinate (5.55), indicating its better anti-HSV-1 activity. The docking and dynamics studies showed montagnetol was stable throughout the 100 ns, having better interactions and docking scores with HSV-1 thymidine kinase than methyl orsellinate, as well as the standard. To understand the mechanism of montagnetol's anti-HSV-1 activity, more research is required, and this could lead to the discovery of new and effective antiviral agents.Communicated by Ramaswamy H. Sarma.

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.

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.