Lichen or Associated Micro-Organism Compounds Are Active against Human Coronaviruses.

(1) Background: Since the emergence of SARS-CoV-2, responsible for the COVID-19 pandemic, efforts have been made to identify antiviral compounds against human coronaviruses. With the aim of increasing the diversity of molecule scaffolds, 42 natural compounds, of which 28 were isolated from lichens and 14 from their associated microorganisms (bacteria and fungi), were screened against human coronavirus HCoV-229E. (2) Methods: Antiviral assays were performed using HCoV-229E in Huh-7 and Huh-7/TMPRSS2 cells and SARS-CoV-2 in a Vero-81-derived clone with a GFP reporter probe. (3) Results: Four lichen compounds, including chloroatranol, emodin, perlatolic acid and vulpinic acid, displayed high activities against HCoV-229E (IC50 = 68.86, 59.25, 16.42 and 14.58 µM, respectively) and no toxicity at active concentrations. Kinetics studies were performed to determine their mode of action. The four compounds were active when added at the replication step. Due to their significant activity, they were further tested on SARS-CoV-2. Perlatolic acid was shown to be active against SARS-CoV-2. (4) Conclusions: Taken together, these results show that lichens are a source of interesting antiviral agents against human coronaviruses. Moreover, perlatolic acid might be further studied for its pan-coronavirus antiviral activity.

A database of high-resolution MS/MS spectra for lichen metabolites.

While analytical techniques in natural products research massively shifted to liquid chromatography-mass spectrometry, lichen chemistry remains reliant on limited analytical methods, Thin Layer Chromatography being the gold standard. To meet the modern standards of metabolomics within lichenochemistry, we announce the publication of an open access MS/MS library with 250 metabolites, coined LDB for Lichen DataBase, providing a comprehensive coverage of lichen chemodiversity. These were donated by the Berlin Garden and Botanical Museum from the collection of Siegfried Huneck to be analyzed by LC-MS/MS. Spectra at individual collision energies were submitted to MetaboLights (https://www.ebi.ac.uk/metabolights/MTBLS999) while merged spectra were uploaded to the GNPS platform (CCMSLIB00004751209 to CCMSLIB00004751517). Technical validation was achieved by dereplicating three lichen extracts using a Molecular Networking approach, revealing the detection of eleven unique molecules that would have been missed without LDB implementation to the GNPS. From a chemist's viewpoint, this database should help streamlining the isolation of formerly unreported metabolites. From a taxonomist perspective, the LDB offers a versatile tool for the chemical profiling of newly reported species.

Lichen butyrolactone derivatives disrupt oral bacterial membrane.

We have previously demonstrated that out of the butyrolactones series synthesized based on the natural lichen metabolite lichesterinic acid, compound (B-13) was the most effective against oral bacteria. However, its antibacterial mechanism is still unknown. In this study, we have investigated its bacterial localization by synthesizing a fluorescently labeled B-13 with NBD while maintaining its antibacterial activity. We showed that this compound binds to Streptococcus gordonii cell surface, as demonstrated by HPLC analysis. By adhering to cell surface, B-13 induced cell wall disruption leading to the release of bacterial constituents and consequently, the death of S. gordonii, a Gram-positive bacterium. A Gram-negative counterpart, Porphyromanas gingivalis, showed also cracked and ruptured cells in the presence of B-13. Besides, we also demonstrated that the analog of B-13, B-12, has also induced disruption of P. gingivalis and S. gordonii. This study revealed that butyrolactones can be considered as potent antibacterial compounds against oral pathogens causing medical complications.

Mycosporine-Like Amino Acids (MAAs) in Time-Series of Lichen Specimens from Natural History Collections.

Mycosporine-like amino acids (MAAs) were quantified in fresh and preserved material of the chlorolichen Dermatocarpon luridum var. luridum (Verrucariaceae/Ascomycota). The analyzed samples represented a time-series of over 150 years. An HPLC coupled with a diode array detector (HPLC-DAD) in hydrophilic interaction liquid chromatography (HILIC) mode method was developed and validated for the quantitative determination of MAAs. We found evidence for substance specific differences in the quality of preservation of two MAAs (mycosporine glutamicol, mycosporine glutaminol) in Natural History Collections. We found no change in average mycosporine glutamicol concentrations over time. Mycosporine glutaminol concentrations instead decreased rapidly with no trace of this substance detectable in collections older than nine years. Our data predict that a screening for MAAs in organism samples from Natural History Collections can deliver results that are comparable to those obtained from fresh collections only for some MAAs (e.g., mycosporine glutamicol). For other MAAs, misleading, biased, or even false negative results will occur as a result of the storage sensitivity of substances such as mycosporine glutaminol. Our study demonstrates the value of pilot studies with time-series based on model taxa with a rich representation in the Natural History Collections.

Tsavoenones A-C: unprecedented polyketides with a 1,7-dioxadispiro[4.0.4.4]tetradecane core from the lichen Parmotrema tsavoense.

New racemic dispiranic polyketides, tsavoenones A (1), B (2) and C (3), having a novel 1,7-dioxadispiro[4.0.4.4]tetradecane scaffold were isolated from the foliose lichen Parmotrema tsavoense. These compounds were structurally elucidated by extensive NMR analyses, comparison between experimental and theoretical 13C NMR data and X-ray crystallography. A putative biosynthetic scenario for the formation of 1-3 from parmosidone D, a meta-depsidone previously isolated from the same lichen material, was proposed. Tested for its cytotoxicity, 1 displayed a moderate activity against human myelogenous leukemia K562 cell line with an IC50 value of 66 µg mL-1.

Antibacterial activities of natural lichen compounds against Streptococcus gordonii and Porphyromonas gingivalis.

The oral bacteria not only infect the mouth and reside there, but also travel through the blood and reach distant body organs. If left untreated, the dental biofilm that can cause destructive inflammation in the oral cavity may result in serious medical complications. In dental biofilm, Streptococcus gordonii, a primary oral colonizer, constitutes the platform on which late pathogenic colonizers like Porphyromonas gingivalis, the causative agent of periodontal diseases, will bind. The aim of this study was to determine the antibacterial activity of eleven natural lichen compounds belonging to different chemical families and spanning from linear into cyclic and aromatic structures to uncover new antibiotics which can fight against the oral bacteria. The compounds were screened by broth microdilution assay. Three compounds were shown to have promising antibacterial activities where the depsidone core with certain functional groups constituted the best compound, psoromic acid, with the lowest MICs=11.72 and 5.86µg/mL against S. gordonii and P. gingivalis, respectively. The compounds screened had promising antibacterial activity which might be attributed to some important functional groups as discussed in our study. The best compounds did not induce the death of gingival epithelial carcinoma cells (Ca9-22). These results introduce new compounds having potent antibacterial activities against oral pathogens causing serious medical complications.

New erythritol derivatives from the fertile form of Roccella montagnei.

Chemical investigation of the methanol extract of the fertile form of Roccella montagnei collected in Vietnam afforded twelve secondary metabolites, including five new montagnetol derivatives, orsellinylmontagnetols A-D and a furanyl derivative together with seven known compounds. Their chemical structures were elucidated by analysis of 1D and 2D NMR and high resolution mass spectroscopic data. The relative stereochemistry of two chiral centers (C-2 and C-3) of orsellinylmontagnetols A and B was elucidated by comparison of their coupling constants and the specific rotation with those reported in the literature while the absolute stereochemistry was determined by the application of a modified Mosher method for the hydroxy group at C-3. The absolute configuration (2R,3S) of the butanetetraol moiety of these compounds is in accordance with that of erythrin, a recognized chemotaxonomic marker of the genus Roccella. Five of these compounds were evaluated for their cytotoxic activities against four cancer cell lines. Only orsellinylmontagnetol A exerted a moderate activity against MCF-7 cell line with an IC50 value of 68.39 ± 3.46 µM.

Design, synthesis and biological evaluation of potential antibacterial butyrolactones.

Novel butyrolactone analogues were designed and synthesized based on the known lichen antibacterial compounds, lichesterinic acids (B-10 and B-11), by substituting different functional groups on the butyrolactone ring trying to enhance its activity. All synthesized butyrolactone analogues were evaluated for their in vitro antibacterial activity against Streptococcus gordonii. Among the derivatives, B-12 and B-13 had the lowest MIC of 9.38µg/mL where they have shown to be stronger bactericidals, by 2-3 times, than the reference antibiotic, doxycycline. These two compounds were then checked for their cytotoxicity against human gingival epithelial cell lines, Ca9-22, and macrophages, THP-1, by MTT and LDH assays which confirmed their safety against the tested cell lines. A preliminary study of the structure-activity relationships unveiled that the functional groups at the C4 position had an important influence on the antibacterial activity. An optimum length of the alkyl chain at the C5 position registered the best antibacterial inhibitory activity however as its length increased the bactericidal effect increased as well. This efficiency was attained by a carboxyl group substitution at the C4 position indicating the important dual role contributed by these two substituents which might be involved in their mechanism of action.

Lichen-derived compounds show potential for central nervous system therapeutics.

BACKGROUND: Natural products from lichens are widely investigated for their biological properties, yet their potential as central nervous system (CNS) therapeutic agents is less explored. PURPOSE: The present study investigated the neuroactive properties of selected lichen compounds (atranorin, perlatolic acid, physodic acid and usnic acid), for their neurotrophic, neurogenic and acetylcholine esterase (AChE) activities. METHODS: Neurotrophic activity (neurite outgrowth) was determined using murine neuroblastoma Neuro2A cells. A MTT assay was performed to assess the cytotoxicity of compounds at optimum neurotrophic activity. Neuro2A cells treated with neurotrophic lichen compounds were used for RT-PCR to evaluate the induction of genes that code for the neurotrophic markers BDNF and NGF. Immunoblotting was used to assess acetyl H3 and H4 levels, the epigenetic markers associated with neurotrophic and/or neurogenic activity. The neurogenic property of the compounds was determined using murine hippocampal primary cultures. AChE inhibition activity was performed using a modified Ellman's esterase method. RESULTS: Lichen compounds atranorin, perlatolic acid, physodic acid and (+)-usnic acid showed neurotrophic activity in a preliminary cell-based screening based on Neuro2A neurite outgrowth. Except for usnic acid, no cytotoxic effects were observed for the two depsides (atranorin and perlatolic acid) and the alkyl depsidone (physodic acid). Perlatolic acid appears to be promising, as it also exhibited AChE inhibition activity and potent proneurogenic activity. The neurotrophic lichen compounds (atranorin, perlatolic acid, physodic acid) modulated the gene expression of BDNF and NGF. In addition, perlatolic acid showed increased protein levels of acetyl H3 and H4 in Neuro2A cells. CONCLUSION: These lichen depsides and depsidones showed neuroactive properties in vitro (Neuro2A cells) and ex vivo (primary neural stem or progenitor cells), suggesting their potential to treat CNS disorders.

UV-protectant metabolites from lichens and their symbiotic partners.

Lichens are structurally complex symbiotic organisms that are exposed to a wide variety of external conditions (extreme temperatures, desiccation, UV radiation, etc.). These poikilohydric organisms have developed various mechanisms of photoprotection, such as light scattering, radiation screening, thermal dissipation, activation of antioxidant defense and macromolecules and membrane repair. These unique organisms produce a vast array of compounds, with more than 1000 secondary metabolites known. An important protective mechanism of lichens is the production of UV screening compounds, such as phenolic compounds (depsidones, depsides, diphenyl ethers), anthraquinones, xanthones or shikimic acid derivatives (calycin, mycosporines, scytonemin). Due to the harmful effects of the UVA wavelengths of sunlight, the search for new sunscreens remains important. We herein propose a review that focuses on the UV protectants from lichens and their symbiotic partners (lichenized fungi, green alga, cyanobacteria). In fact, lichens produce unique and/or efficient UV filters such as depsidones (lobaric acid, pannarin, etc.), depsides (atranorin, gyrophoric acid, etc.), diphenyl ethers (epiphorellic acids, buellin), bisxanthones (secalonic acids, etc.), mycosporines and MAAs, scytonemin along with classical pigments (melanin, carotenoids). We propose to classify these compounds with regard to their chemical structures and review the physicochemical properties that act as UV filters. While the most abundant lichen polyfunctionalized aromatic compounds, belonging to orsellinic derivatives, are UVB screens, these organisms produce strong UVA filters, e.g., calycin (pulvinic acid derivatives), bisxanthones (secalonic acids), scytonemin or mycosporines and MAAs with the latter ones exhibiting attractive properties as photoprotectants.

Characterization and identification of mycosporines-like compounds in cyanolichens. Isolation of mycosporine hydroxyglutamicol from Nephroma laevigatum Ach.

Mycosporine-like compounds, comprising mycosporines and mycosporine-like amino acids (MAAs) are UV protecting secondary metabolites described in organisms such as fungi, algae, cyanobacteria or animals. Lichens however, were only poorly investigated for such constituents so far. Here, a method for the characterization of mycosporines and MAAs in purified aqueous extracts, involving HPTLC coupled to spectrophotodensitometry, HPLC-DAD-MS(n) and UPLC-HRMS analysis, is described. This optimized protocol was validated on three algae and one cyanolichen containing known MAAs and mycosporines, and then applied to 18 cyanolichen species. Analyses revealed the presence of five already described mycosporine-like compounds in the investigated species, including mycosporine serinol in Lichina and Peltigera species and mycosporine glutamicol in Degelia plumbea. Apart from that, eight unknown mycosporine-like compounds were detected and tentatively characterized on the basis of their DAD spectra and their MS(n) and HRMS data: two in the alga Porphyra dioica and six in cyanolichen species belonging to the genera Degelia, Nephroma and Stereocaulon. From Nephroma laevigatum, the mycosporine hydroxyglutamicol was preparatively isolated and identified through HRMS, 1D and 2D NMR spectroscopic data. The optimized analytical protocol allowed the characterization of mycosporine-like compounds in small amounts of material and confirmed the potential of cyanolichens as a source of mycosporine compounds. It should also be applicable to investigate lichen species with green algae photobionts for mycosporine-like compounds.

A novel aryl-hydrazide from the marine lichen Lichina pygmaea: isolation, synthesis of derivatives, and cytotoxicity assays.

A new aryl-hydrazide l-glutamic acid derivative, pygmeine (3), was isolated from a methanolic extract of Lichina pygmaea, a marine lichen. Synthetic derivatives obtained via a two-step coupling of l-glutamic acid with phenylhydrazine moieties were useful to elucidate the structure of 3 and to carry out biological assays. Thus, the cytotoxicity of the ortho-, meta-, and para-hydroxyl isomers along with their respective benzyl intermediates, and a natural methoxylated analog, were evaluated on murine and human melanoma cells (B16, A375). The para-hydroxyl isomer 6 was found to be the most active (IC(50)=1.6 microM) on B16 cells.

Multiple dual-mode centrifugal partition chromatography as an efficient method for the purification of a mycosporine from a crude methanolic extract of Lichina pygmaea.

Centrifugal partition chromatography method was applied to the separation and purification of a crude methanolic extract of a cyanobacterial lichen, Lichina pygmaea. A multiple dual-mode was used to separate two compounds of interest, namely mycosporine-serinol and a glutamic acid derivative. These compounds are described here for the first time in a lichen. Their structures were identified by UV, IR, ESI-MS, (1)H NMR, (13)C NMR, and 2D NMR.

Preparation and characterization of copper(II) and nickel(II) complexes of a new chiral salen ligand derived from (+)-usnic acid.

Chiral copper(II) and nickel(II) complexes were obtained after reaction of diacetate salts with a new chiral salen ligand derived from (+)-usnic acid.