Lichen-forming fungi are unique organisms, producing biologically active metabolites with a great variety of effects, including antibiotic, antimycobacterial, antiviral, anti-inflammatory, analgesic, antipyretic, antiproliferative and cytotoxic activities. However, only very limited numbers of lichen substances have been screened for their biological activities and their therapeutic potential in medicine. This is certainly due to the difficulties encountered in identification of the species, collection of bulk quantities, and the isolation of pure substances for structure determination and testing activity. Recently, possibilities for bypassing some of these former difficulties have arisen by the introduction of new techniques. This includes axenic cultivation for production of the genuine compounds or new ones, extraction of focused compounds, or synthesis of natural products or their derivatives for testing. Utilizing these new opportunities, the discovery of novel active metabolites, which could serve as lead compounds, is significantly facilitated. At the same time, the evolution of secondary metabolite patterns is studied using phylogenetic approaches. Yet, the genetic background of the complex chemical patterns is poorly understood. The scattered occurrence of some compounds suggests that their production evolved either in parallel or that ancient biosynthetic pathways are abandoned in many lineages. At least, studies on polyketide synthase genes from different lichen groups suggest a high level of gene paralogy. In this context, clades of orthologous polyketide synthase genes, which are often shared with distantly related non-lichenized fungi, can roughly be identified by their sequence similarity and their similar patterns of substitution rates. The functional assignment of paralogs is nevertheless difficult and reasonable only in a few cases. A global approach of the lichen metabolomic features appears to be essential in developing new and viable biotechnological processes which could afford suitable amounts of unique lichen compounds.

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.

Considering the importance of ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-tandem mass spectrometry (UPLC-ESI-QTOF-MS/MS) hyphenated techniques for analysis of secondary metabolites from crude extracts, the present study was aimed at identification of secondary metabolites in acetone extract of the lichen Usnea longissima. From our study, 19 compounds were tentatively identified through comparison of exact molecular masses from their MS/MS spectra, mass fragmentation studies and comparison with literature data. In addition, potent cytotoxic activity of U. longissima extract prompted us to isolate four compounds, 18R-hydroxy-dihydroalloprotolichesterinic acid (19), neuropogolic acid (20), barbatic acid (21), and usnic acid (22) from this extract which were adequately identified through mass spectrometry and NMR spectroscopy. All four compounds displayed cytotoxic activity. Barbatic acid (21) manifested doxorubicin equivalent activity against A549 lung cancer cell line with IC₅₀ of 1.78 µM and strong G0/G1 accumulation of cells. Poly ADP-ribose polymerase (PARP) cleavage confirmed that it induced cytotoxic activity via apoptosis. Finally, our work has discerned the depside, barbatic acid (21) from crude extract as a candidate anti-cancer molecule, which induces cell death by stepping up apoptosis.