Rugulosin A (RugA), a bis-anthraquinone isolated from the endolichenic fungus Coniochaeta velutina, was evaluated for its antifungal activity against the phytopathogenic fungi Botrytis cinerea and Alternaria alternata. RugA inhibited mycelial growth of both pathogens, showing greater strength against Botrytis cinerea (IC50 = 2.5 μM) than against Alternaria alternata (IC50 = 10 μM). Nevertheless, concerning spore germination inhibition, RugA was slightly more efficient on Alternaria than on Botrytis, with a MIC of 75 μM for Alternaria alternata and 100 μM for Botrytis cinerea. Treatment with 25 μM RugA reduced the expression of genes involved in spore germination and pathogenicity of Botrytis cinerea. Confocal microscopy using propidium iodide, DCFH-DA, and 2-NBDG revealed that RugA disrupted membrane integrity, induced oxidative stress, and impaired sugar uptake, ultimately leading to fungal cell death. Molecular docking analyses further indicated a strong interaction between RugA and NADPH-cytochrome P450 reductase, with a binding affinity of −11.2 kcal/mol, which was associated with reduced enzyme activity and may contribute to the observed antifungal effects. Moreover, the expression of some grapevine defense genes, such as PR1, PR3, PR4, STS, HSR1, and EDS1, involved in pathogenicity, was not induced on infected leaves treated with RugA, suggesting a protective effect of RugA. Furthermore, 100 μM RugA spraying did not show any toxicity to housefly maggots and grapevine plantlets. Taken together, these data suggested that RugA could be a promising candidate for combating phytopathogenic fungi for further applications in agriculture.

Five previously undescribed diterpenoids (1-5) along with four known ones (6-9) were isolated from the roots of Croton crocodilorum. Most notably, the four ent-3,4-seco-atisane diterpenoids (2-5) biosynthesized from the ent-atisane diterpenoid (1) were crotogoudin derivatives. Their structures were elucidated by spectroscopic data including HRESIMS and extensive 1D and 2D NMR investigations. The absolute configuration of compounds (-)-1, (-)-3 and (-)-6 was determined by vibrational circular dichroism (VCD) and Raman optical activity (ROA) spectroscopies along with density functional theory calculations. Their plausible biosynthetic pathway was proposed. All isolated diterpenoids were evaluated for their antiproliferative activity against sensitive and resistant human colorectal cancer cells (HCT116 and HT-29). Compounds 3, 5, 6 and 7 displayed more potent activities than the two positive controls (5 FU and irinotecan) against the two cancer cell lines.

Specific natural compounds of Humulus lupulus L. (hop cones) have significant applications both in food and pharmaceutical industries. The effectiveness of these applications depends on the extraction methods used, which can vary in the yield and metabolite profile of the extract. This study focuses on the development of green solvent extraction methods in order to adapt the process to industrial-scale extraction in other plant species. Four extraction techniques were employed, including maceration (simple maceration (SM) and triphasic-SE), ultrasound (UAE), high pressure (PLE), microwaves (MAE). Two of them were also combined (UAE+SM). Ethanol and a combination of ethanol and water were mostly used. Triphasic extraction was performed with classical solvents. The results were compared in term of yield, total assays (polyphenols, flavonoids, and chlorophylls), metabolite profile, LC-MS/MS quantification, and molecular networking. UAE led to high yield with reduced extraction time compared to maceration, and this result was further amplified with pressure-assisted extraction (PLE) with a 3- and 6-fold increase in yield and time reduction, respectively. The yields were correlated with higher quantities of molecules of interest, such as alpha/(3 acids, polyphenols, and flavonoids. For instance, the MAE and PLE techniques provided high content of flavonoids and polyphenols (2 and 3-fold), xanthohumol (6 and 4fold) compared to SE. The use of alternative extraction methods alone or coupled significantly improved the yields and contents. LC-MS/MS allowed for the monitoring of various metabolite concentrations, which were also found in molecular networks, and that facilitating the selection of the most appropriate technique. A descriptive statistical analysis was used to provide a global vision between the different extractive methods and determine the best experimental conditions. Among the various treatments tested, the global desirabilities from 65.5 % and 77.4% were achieved with PLE followed by UAE-B+SM (64.5 %). They emerged as the most effective approach providing a balance between mass yields, content and concentration of metabolites of interest.

Lichen substances have been first described in the 1870s, and around 10 000 compounds have been isolated and characterized. Most of them have been evaluated for their activity on planktonic microorganisms (bacteria and fungi). More recently, microorganisms colonizing the lichen thallus have been isolated and identified using DNA sequencing, giving access to a wide diversity of culturable microorganisms. The increasing research in lichen-associated microbiomes in recent years has emphasized a wide range of metabolites as a potential source of bioactive compounds. In parallel, humans are facing microbial resistance to conventional antimicrobial drugs. One of the reasons is the biofilm lifestyle of microorganisms. Indeed, the aggregation of microbial communities inside biofilms is now well known and characterized, and some possible ways to fight and destroy biofilms are identified (quorum sensing inhibitors, etc.). The present review aims to summarize the anti-biofilm potential of lichen metabolites and those from their associated microorganisms (bacteria and/or fungi). Are the metabolites isolated from lichens and their associated fungi displaying any anti-biofilm activity? This literature synthesis highlights the metabolites of interest as new anti-biofilm drugs and shows the lack of current biological research dealing with biofilm and lichen metabolites. Acetone and ethyl acetate extracts are the most studied sources of anti-biofilm agents. Only two lichen metabolites, usnic acid and evernic acid, have been evaluated both as antifungal and antibacterial biofilm compounds. Terpenoids from lichens are still poorly explored for this activity.

The endolichenic fungus Aspergillus sp. was isolated from the lichen Xanthoparmelia conspersa harvested in France. Aspergillus sp. was grown on a solid culture medium to ensure the large-scale production of the fungus with a sufficient mass of secondary metabolites. The molecular network analysis of extracts and subfractions enabled the annotation of 22 molecules, guiding the purification process. The EtOAc extract displayed an antiproliferative activity of 3.2 ± 0.4 µg/mL at 48 h against human colorectal cancer cells (HT-29) and no toxicity at 30 µg/mL against human triple-negative breast cancer (TNBC) cells (MDA-MB-231) and human embryonic kidney (HEK293) non-cancerous cells. Among the five prenylated compounds isolated, of which four are echinulin derivatives, compounds 1 and 2 showed the most important activity, with IC50 values of 1.73 µM and 8.8 µM, respectively, against HT-29 cells.

(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.

Recently, the study of the interactions within a microcosm between hosts and their associated microbial communities drew an unprecedented interest arising from the holobiont concept. Lichens, a symbiotic association between a fungus and an alga, are redefined as complex ecosystems considering the tremendous array of associated microorganisms that satisfy this concept. The present study focuses on the diversity of the microbiota associated with the seashore located lichen Rhizocarpon geographicum, recovered by different culture-dependent methods. Samples harvested from two sites allowed the isolation and the molecular identification of 68 fungal isolates distributed in 43 phylogenetic groups, 15 bacterial isolates distributed in five taxonomic groups and three microalgae belonging to two species. Moreover, for 12 fungal isolates belonging to 10 different taxa, the genus was not described in GenBank. These fungal species have never been sequenced or described and therefore non-studied. All these findings highlight the novel and high diversity of the microflora associated with R. geographicum. While many species disappear every day, this work suggests that coastal and wild environments still contain an unrevealed variety to offer and that lichens constitute a great reservoir of new microbial taxa which can be recovered by multiplying the culture-dependent techniques.

Endolichenic microorganisms represent a new source of bioactive natural compounds. Lichens, resulting from a symbiotic association between algae or cyanobacteria and fungi, constitute an original ecological niche for these microorganisms. Endolichenic fungi inhabiting inside the lichen thallus have been isolated and characterized. By cultivation on three different culture media, endolichenic fungi gave rise to a wide diversity of bioactive metabolites. A total of 38 extracts were screened for their anti-maturation effect on Candida albicans biofilms. The 10 most active ones, inducing at least 50% inhibition, were tested against 24 h preformed biofilms of C. albicans, using a reference strain and clinical isolates. The global molecular network was associated to bioactivity data in order to identify and priorize active natural product families. The MS-targeted isolation led to the identification of new oxygenated fatty acid in Preussia persica endowed with an interesting anti-biofilm activity against C. albicans yeasts.

While depsidones, depsides or dibenzofuran-like compounds dominate the chemical composition of lichens, the cyanolichen Nephroma laevigatum affords a diversity of quinoid pigments represented by chlorinated anthraquinones derived from emodin and new bianthrones resulting from the homo- or heterodimerization of monomers. Bianthrones were pointed out from the dichloromethane extract by MS/MS-based molecular networking, then isolated and characterized on the basis of extensive spectroscopic analyzes and GIAO NMR shift calculation followed by CP3 analyzes.

Lichens, due to their symbiotic nature (association between fungi and algae), constitute a chemical factory of original compounds. Polyphenolic compounds (depsides and depsidones) are the main constituents of lichens and are exclusively biosynthesized by these organisms. A panel of 11 polyphenols was evaluated for their anti-biofilm activity against Candida albicans biofilms on the maturation phase (anti-maturation) (MMIC50) as well as on preformed 24-h-old biofilm (anti-biofilm) (MBIC50) using the XTT assay. Minimum inhibitory concentrations of compounds (MICs) against C. albicans planktonic yeast were also determined using a broth microdilution method. While none of the tested compounds were active against planktonic cells (IC50 > 100 µg/ml), three depsides slowed the biofilm maturation (MMIC50 ≤12.5 µg/ml after 48 h of contact with Candida cells). Evernic acid was able to both slow the maturation and reduce the already formed biofilms with MBIC50 ≤12.5 µg/ml after 48 h of contact with the biofilm. This compound shows a weak toxicity against HeLa cells (22%) at the minimal active concentration and no hemolytic activity at 100 µg/ml. Microscopic observations of evernic acid and optimization of its solubility were performed to further study this compound. This work confirmed the anti-biofilm potential of depsides, especially evernic acid, and allows to establish the structure–activity relationships to better explain the anti-biofilm potential of these compounds.

Acetone extracts of the two common epiphytes lichens Usnea florida and Flavoparmelia caperata have been evaluated for their antimicrobial activities against Staphylococcus aureus, Candida albicans and Aspergillus brasiliensis. The dibenzofuran derivative (+)-usnic acid (1) was the main metabolite in these two species. Thamnolic (5), evernic (6), physodic (7) and 3-hydroxyphysodic acids (8) were isolated from U. florida, as well as 5,7-dihydroxy-6-methylphtalide (2) which was newly identified in this Genus. Protocetraric (3) and caperatic acids (4) and ergosterol peroxide (9) are usually biosynthezised by F. caperata. Antibacterial activity was determined for the four main compounds against Staphylococcus aureus using bioautography and broth dilution method. Minimal inhibitory concentrations of usnic acid, caperatic acid and protocetraric acid were comprised between 7.25 and 12.5 µg/mL.

This Data Descriptor announces the submission to public repositories of the monoterpene indole alkaloid database (MIADB), a cumulative collection of 172 tandem mass spectrometry (MS/MS) spectra from multiple research projects conducted in eight natural product chemistry laboratories since the 1960s. All data have been annotated and organized to promote reuse by the community. Being a unique collection of these complex natural products, these data can be used to guide the dereplication and targeting of new related monoterpene indole alkaloids within complex mixtures when applying computer-based approaches, such as molecular networking. Each spectrum has its own accession number from CCMSLIB00004679916 to CCMSLIB00004680087 on the GNPS. The MIADB is available for download from MetaboLights under the identifier: MTBLS142 ( https://www.ebi.ac.uk/metabolights/MTBLS142 ).

Aims The objective of this study was to explore the diversity of endolichenic fungi from Nephroma laevigatum and to investigate their antiproliferative and antibiofilm potential. Methods and Results Forty-six isolates were obtained and identified by DNA barcoding. They belonged to genera Nemania, Daldinia, Peziza and Coniochaeta. Six strains belonging to the most represented species were selected and tested for their antiproliferative and antibiofilm activities. Extracts were analysed by reversed-phase HPLC. Activities against fungal and bacterial biofilm were evaluated using tetrazolium salt (XTT) assay and crystal violet assay respectively. Antiproliferative responses of extracts were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induction by two extracts was observed in two cell lines (HT-29 and PC-3) via morphological changes, pro-apoptotic and anti-apoptotic proteins analysis (Western blotting) and DNA fragmentation. Four extracts displayed activities against Candida albicans biofilm with IC50 values ranging from 25 to 200 mu g ml(-1). All extracts were inactive against Staphylococcus aureus and Pseudomonas aeruginosa biofilms. The most active isolates against human colorectal (HT-29 and HCT116) and prostate (PC-3 and DU145) cancer cell lines were Nemania serpens (NL08) and Nemania aenea var. aureolatum (NL38) with IC50 values ranging from 13 to 39 mu g ml(-1). These extracts induced an apoptotic process through activation of caspases 8 and 3, poly(ADP-ribose) polymerase cleavage and DNA fragmentation. Conclusions Selected crude fungal extracts have antiproliferative and antibiofilm activities. Data suggest that this antipoliferative effect is due to apoptosis process. This is the first report showing the effects of endolichenic fungi from N. laevigatum. Significance and Impact of the Study This study highlights the therapeutic potential of endolichenic fungi metabolites as sources for drug discovery programmes.

Fungal communities associated to three epiphytic lichens active against Candida, were investigated using culture-based methods We hypothetized that associated fungi would contribute to lichens activities. The ability of specific fungi to grow inside or outside lichens was investigated. To detect biogenesis pathways involved in the production of secondary metabolites, genes coding for nonribosomal peptide synthetase (NRPS) and polyketide synthase I (PKS I) were screened by PCR from fungal DNA extracts. Both endo and epilichenic communities were isolated from two fructicose (Evernia prunastri and Ramalina fastigiata) and one foliose (Pleurosticta acetabulum) lichens. A total of 86 endolichenic and 114 epilichenic isolates were obtained, corresponding to 18 and 24 phylogenetic groups respectively suggesting a wide diversity of fungi. The communities and the species richness were distinct between the three lichens which hosted potentially new fungal species. Additionally, the endo- and epilichenic communities differed in their composition: Sordariomycetes were particularly abundant among endolichenic fungi and Dothideomycetes among epilichenic fungi. Only a few fungi colonized both habitats, such as S. fimicola, Cladosporium sp1 and Botrytis cinerea. Interestingly, Nemania serpens (with several genotypes) was the most abundant endolichenic fungus (53% of isolates) and was shared by the three lichens. Finally, 12 out of 36 phylogenetic groups revealed the presence of genes coding for nonribosomal peptide synthetase (NRPs) and polyketide synthase I (PKS I). This study shows that common lichens are reservoirs of diverse fungal communities, which could potentially contribute to global activity of the lichen and, therefore, deserve to be isolated for further chemical studies.

The composition of sapwood, transition zone and heartwood extracts from Pseudotsuga menziesii was studied using LC/ESI–MS/MS. Based on retention times, and MS fragmentation patterns, 40 phenolic compounds, 11 terpenes and 7 fatty and organic acids were identified or accordingly characterized. The flavonoids taxifolin-O-hexoside and the corresponding aglycon taxifolin, dihydrokaempferol, pinocembrin, pinobanksin, quercetin and flavan-3-ols, catechin and epicatechin were the main compounds detected in wood collected in the fall. Taxifolin derivatives were identified as monomers, dimers and flavonolignans. Comparative metabolite profiling of the three zones showed differences in metabolite distribution. Flavonoids were distributed in the 3 zones. Lignans, flavonolignans, phenolic acid derivatives and terpenes accumulated mainly in the transition zone while tannins were detected only in sapwood. Fatty and organic acids were present in sapwood and the transition zone. Multiple Reaction Monitoring (MRM) was used for the quantification of main metabolites of extracts in order to observe seasonal variations in the wood. The relative quantity of 35 selected compounds in each zone was discussed. The lowest metabolite content was usually observed in winter whereas flavonoids were found in high quantities in heartwood either in spring or autumn.

Candida albicans is a commensal coloniser of the human gastrointestinal tract and an opportunistic pathogen, especially thanks to its capacity to form biofilms. This lifestyle is frequently involved in infections and increases the yeast resistance to antimicrobials and immune defenses. In this context, 38 lichen acetone extracts have been prepared and evaluated for their activity against C. albicans planktonic and sessile cells. Minimum inhibitory concentrations of extracts (MICs) were determined using the broth microdilution method. Anti-biofilm activity was evaluated using tetrazolium salt (XTT) assay as the ability to inhibit the maturation phase (anti-maturation) or to eradicate a preformed 24 h old biofilm (anti-biofilm). While none of the extracts were active against planktonic cells, biofilm maturation was limited by 11 of the tested extracts. Seven extracts displayed both anti-maturation and anti-biofilm activities (half maximal inhibitory concentrations IC50_mat and IC50_biof ≤ 100 µg/mL); Evernia prunastri and Ramalina fastigiata were the most promising lichens (IC50_mat < 4 µg/mL and IC50_biof < 10 µg/mL). Chemical profiles of the active extracts performed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) have been analyzed. Depsides, which were present in large amounts in the most active extracts, could be involved in anti-biofilm activities. This work confirmed that lichens represent a reservoir of compounds with anti-biofilm potential.