Δ-Keto-acid/hydroxy-lactone isomerization in some lichen depsides, depsidones and diphenyl ethers.

In some compounds in lichens, the carboxylic acid is ortho-substituted by an 2-oxoalkyl chain. This particular structure induces the existence of δ-keto-acid ka or hydroxy-lactone hl isomers, clearly identified by their NMR data and chemical properties, such as dehydration, methylation and behaviour in thermal conditions. Internal hydrogen bonding between the carboxylic acid and substituent in the ortho' position is proposed as an isomerization modulator: an H-bond acceptor (OCH3) leads to ka isomers, whereas hl isomers are obtained with an H-bond donor (OH).

Eremoxylarins D-J, Antibacterial Eremophilane Sesquiterpenes Discovered from an Endolichenic Strain of Xylaria hypoxylon.

An endolichenic strain of the Ascomycetaceous Xylaria hypoxylon, cultivated alone or in coculture with another endolichenic fungus Dendrothyrium variisporum, produced seven new bioactive eremophilane sesquiterpenes eremoxylarins D-J (1-7). The isolated compounds disclosed a high similarity with the eremophilane core of the bioactive integric acid, and structures were elucidated by 1D and 2D NMR spectra and electronic circular dichroism (ECD) analyses. Eremoxylarins D, F, G, and I showed a selective activity against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus with minimum inhibitory concentration (MIC) values between 0.39 and 12.5 µg/mL. Eremoxylarin I, the most antibacterial active sesquiterpene, was also active against HCoV-229E at a concentration nontoxic to the hepatoma Huh-7 cell line with an 50% inhibitory concentration (IC50) of 18.1 µM and a 50% cytotoxic concentration (CC50) of 46.6 µM.

Efficiency and selectivity of ionic liquids in microwave-assisted extraction of major lichen phenolic compounds: a scalable process with recycling of ionic liquids.

INTRODUCTION: Pseudevernia furfuracea, a lichen used classically for cosmetic applications, contains interesting metabolites possessing antimicrobial and anti-inflammatory or antioxidant properties. OBJECTIVES: Ionic liquid combined to microwave-assisted extraction (IL-MAE) was successfully applied for metabolites extraction from Pseudevernia furfuracea. MATERIALS AND METHODS: Three imidazolium and pyridinium-based ionic liquids (ILs): 1,3-dimethylimidazolium methylsulphate [C1 C1 Im][MeSO4 ], 1-ethyl-3-methylimidazolium ethylsulphate [C2 C1 Im][EtSO4 ], and N-ethylpyridinium ethylsulphate [C2 Py][EtSO4 ] were assessed for this process. The efficiency of the extraction method was evaluated using thin-layer chromatography (TLC) coupled to a Camag® spectrophotodensitometer and using high-performance liquid chromatography (HPLC) analysis. RESULTS: ILs under MAE showed extraction time efficiency (15 min vs. 24 h for conventional heating) and high selectivity in extracting the targeted metabolites: atranorin (AT), methyl-ß-orcinol carboxylate (MOC), fumarprotocetraric acid (Fum. Ac.), and physodic acid (Phys. Ac.) despite the increased degradation of AT under MAE. We showed a tunable selectivity of ILs towards extracting metabolites by changing anion or cation due to the modification of the interaction between the IL and the metabolites. While [C2 Py][EtSO4 ] was the most efficient IL and could extract all the targeted metabolites, [C2 C1 Im][EtSO4 ] was the most selective. It fully extracted AT and partially Fum. Ac. Moreover, the lichen prepared by mixing procedure provided AT and Fum. Ac. more than the milled one. A 100 times scale-up extraction was successfully performed on mixed samples with full IL recycling after back extraction. CONCLUSION: IL-MAE is reliable for lichen metabolites extraction. The method is reproducible, scalable, with possible IL recycling, opening the door for potential industrial applications.

tert-Butylphenolic Derivatives from Paenibacillus odorifer-A Case of Bioconversion.

Two compounds (1) and (2) containing tert-butylphenol groups were, for the first time, produced during the culture of Paenibacillus odorifer, a bacterial strain associated with the crustose lichen, Rhizocarpon geographicum. Their entire structures were identified by one-dimensional (1D) and two-dimensional (2D) NMR and high-resolution electrospray ionisation mass spectrometry (HRESIMS) spectroscopic analyses. Among them, Compound 1 exhibited significant cytotoxicity against B16 murine melanoma and HaCaT human keratinocyte cell lines with micromolar half maximal inhibitory concentration (IC50) values. Furthermore, after supplementation studies, a putative biosynthesis pathway was proposed for Compound 1 throughout a bioconversion by this bacterial strain of butylated hydroxyanisole (BHA), an antioxidant polymer additive.

Isolation and Structure Identification of Novel Brominated Diketopiperazines from Nocardia ignorata-A Lichen-Associated Actinobacterium.

Actinobacteria are well known for their potential in biotechnology and their production of metabolites of interest. Lichens are a promising source of new bacterial strains, especially Actinobacteria, which afford a broad chemical diversity. In this context, the culture medium of the actinobacterium Nocardia ignorata, isolated from the terrestrial lichen Collema auriforme, was studied. The strain was cultivated in a BioFlo 115 bioreactor, and the culture medium was extracted using an XAD7HP resin. Five known diketopiperazines: cyclo (l-Pro-l-OMet) (1), cyclo (l-Pro-l-Tyr) (2), cyclo (d-Pro-l-Tyr) (3), cyclo (l-Pro-l-Val) (4), cyclo (l-Pro-l-Leu) (5), and one auxin derivative: indole-carboxaldehyde (8) were isolated, along with two new brominated diketopiperazines: cyclo (d-Pro-l-Br-Tyr) (6) and cyclo (l-Pro-l-Br-Tyr) (7). Structure elucidation was performed using HRMS and 1D and 2D NMR analysis, and the synthesis of compounds 6 and 7 was carried out in order to confirm their structure.

Dibenzofurans and derivatives from lichens and ascomycetes.

Covering: up to 2016.When looking for dibenzofuran in the biochemical databases, most papers and reviews deal with pollutants and polychlorinated dibenzofurans like dioxins. But dibenzofurans are also biosynthetized by a wide diversity of organisms in nature. Even if dibenzofurans from natural sources represent a small class of secondary metabolites, compared to flavonoids, xanthones or terpenoids, they are often endowed with interesting biological properties which have been recently described. This review provides an update on papers describing dibenzofurans from lichens, ascomycetes and cultured mycobionts. Other sources, such as basidiomycetes, myxomycetes or plants produce sporadically interesting dibenzofurans in terms of structures and activities.

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.

Depsidones from Lichens as Natural Product Inhibitors of M-Phase Phosphoprotein 1, a Human Kinesin Required for Cytokinesis.

M-Phase Phosphoprotein 1 (MPP1), a microtubule plus end directed kinesin, is required for the completion of cytokinesis. Previous studies have shown that MPP1 is upregulated in various types of bladder cancer. This article describes inhibitor screening leading to the identification of a new class of natural product inhibitors of MPP1. Two compounds with structural similarity, norlobaridone (1) and physodic acid (2), were found to inhibit MPP1. Physodic acid is not competitive with ATP, indicating the presence of an allosteric inhibitor-binding pocket. Initial drug-like property screening indicates that physodic acid is more soluble than norlobaridone and has more favorable lipophilicity. However, both suffer from high clearance in human microsomal stability assays mediated by the lability of the lactone ring as well as hydroxylation of the alkyl chains as shown by metabolite identification studies. In cell-based assays physodic acid is a weak inhibitor with EC50 values of about 30 µM in a range of tumor cell lines. The two depsidones identified and characterized here could be used for future improvement of their activity against MPP1 and will be useful chemical probes for studying this unique molecular motor in more depth.

Lichens as natural sources of biotechnologically relevant bacteria.

The search for microorganisms from novel sources and in particular microbial symbioses represents a promising approach in biotechnology. In this context, lichens have increasingly become a subject of research in microbial biotechnology, particularly after the recognition that a diverse community of bacteria other than cyanobacteria is an additional partner to the traditionally recognized algae-fungus mutualism. Here, we review recent studies using culture-dependent as well as culture-independent approaches showing that lichens can harbor diverse bacterial families known for the production of compounds of biotechnological interest and that several microorganisms isolated from lichens, in particular Actinobacteria and Cyanobacteria, can produce a number of bioactive compounds, many of them with biotechnological potential.

Review - Lichen-Associated Bacteria as a Hot Spot of Chemodiversity: Focus on Uncialamycin, a Promising Compound for Future Medicinal Applications.

This review presents the state of knowledge on the medicinal potential of bacteria associated with lichens. In fact, besides the classical symbiotic partners (photobiont and mycobiont) forming the lichen thallus, associated bacteria have been recently described as a third partner. Various studies demonstrated the diversity of these communities with a predominance of Alphaproteobacteria. Bacterial groups more relevant for secondary metabolite synthesis have also been revealed. This article summarizes studies reporting the abilities of these communities to produce metabolites with relevant bioactivities. The biotechnological interest of these bacteria for drug discovery is highlighted regarding the production of compounds with therapeutic potential. Special focus is given to the synthesis of the most promising compound, uncialamycin, a potent enediyne isolated from a Streptomyces sp. associated with Cladonia uncialis.

Qualitative and spatial metabolite profiling of lichens by a LC-MS approach combined with optimised extraction.

INTRODUCTION: Lichens are self-sustaining partnerships comprising fungi as shape-forming partners for their enclosed symbiotic algae. They produce a tremendous diversity of metabolites (1050 metabolites described so far). OBJECTIVES: A comparison of metabolic profiles in nine lichen species belonging to three genera (Lichina, Collema and Roccella) by using an optimised extraction protocol, determination of the fragmentation pathway and the in situ localisation for major compounds in Roccella species. METHODS: Chemical analysis was performed using a complementary study combining a Taguchi experimental design with qualitative analysis by high-performance liquid chromatography coupled with mass spectrometry techniques. RESULTS: Optimal conditions to obtain the best total extraction yield were determined as follows: mortar grinding to a fine powder, two successive extractions, solid:liquid ratio (2:60) and 700 rpm stirring. Qualitative analysis of the metabolite profiling of these nine species extracted with the optimised method was corroborated using MS and MS/MS approaches. Nine main compounds were identified: 1 ß-orcinol, 2 orsellinic acid, 3 putative choline sulphate, 4 roccellic acid, 5 montagnetol, 6 lecanoric acid, 7 erythrin, 8 lepraric acid and 9 acetylportentol, and several other compounds were reported. Identification was performed using the m/z ratio, fragmentation pathway and/or after isolation by NMR analysis. The variation of the metabolite profile in differently organised parts of two Roccella species suggests a specific role of major compounds in developmental stages of this symbiotic association. CONCLUSION: Metabolic profiles represent specific chemical species and depend on the extraction conditions, the kind of the photobiont partner and the in situ localisation of major compounds.

A lupeol derivative and other isolates with antiplasmodial activity from the stem root of Rauvolfia mannii Stapf. (Apocynaceae).

Rauvolfia mannii is a plant from western and eastern areas of African continent and is widely used in folk medicine for the treatment of various diseases including malaria. Herein, one previously undescribed acylated triterpene (1), together with five already published natural products (2-6) were removed from its roots. The chemical structures of these compounds were determined by spectroscopic and spectrometric means (NMR, HRESIMS, IR and UV). In addition to the isolated triterpenoids, components 5 and 6 are also newly reported from the genus Rauvolfia. Moreover, some constituents were further tested against the chloroquine-sensitive strain of P. falciparum (3D7). It has been found that 3 and 4 showed a moderate antiplasmodial activity with IC50 values of 46.25 and 39.79 µM respectively.

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.

Gold-mediated synthesis and functionalization of chiral halopyridones.

A rapid and efficient one-step halopyridone synthesis has been developed based on gold-catalyzed cyclization of ß-amino-ynone intermediates and halodeauration process.

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.

When the nine-membered enediynes play hide and seek.

The lack of stability of the 9-membered enediynes not associated with an apoprotein may explain the low number of isolated natural compounds containing this core. To overcome such a problem, particular attention should be paid during the process of extraction and isolation of secondary metabolites, especially from microorganisms such as actinomycetes in order to identify the non-cycloaromatized derivatives.

Microbial community associated with the crustose lichen Rhizocarpon geographicum L. (DC.) living on oceanic seashore: A large source of diversity revealed by using multiple isolation methods.

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.

Volatile Organic Compounds from a Lichen-Associated Bacterium, Paenibacillus etheri, Interact with Plant-Parasitic Cyst Nematodes.

Healthy food is one of the major challenges to develop in this century. Plant-parasitic nematodes cause significant damage to many crops worldwide and till now, the use of chemical nematicides is the main means to control their populations. These chemical products must be replaced by more environmental-friendly control methods. Biocontrol methods seem to be one promising option, and the number of biopesticides derived from living organisms has increased in the last decades. To develop new plant protection products, we have decided to combine our skills in natural products chemistry and nematology and to focus on the lichen microecosystem as underexploited ecological niches of microorganisms. We present herein the potential of lichen-associated bacterial suspensions from Paenibacillus etheri as nematicides against the beet cyst nematode Heterodera schachtii and the potato cyst nematode Globodera pallida, in particular the effects of volatile organic compounds (VOCs) produced by the bacteria. A solid phase micro-extraction method associated to gas chromatography-mass spectrometry analysis of 14 day cultures was used to analyze these VOCs in order to identify the main produced compounds (isoamyl acetate and 2-phenylethyl acetate) and to evaluate them on the nematodes.

Rhizocarpon geographicum Lichen Discloses a Highly Diversified Microbiota Carrying Antibiotic Resistance and Persistent Organic Pollutant Tolerance.

As rock inhabitants, lichens are exposed to extreme and fluctuating abiotic conditions associated with poor sources of nutriments. These extreme conditions confer to lichens the unique ability to develop protective mechanisms. Consequently, lichen-associated microbes disclose highly versatile lifestyles and ecological plasticity, enabling them to withstand extreme environments. Because of their ability to grow in poor and extreme habitats, bacteria associated with lichens can tolerate a wide range of pollutants, and they are known to produce antimicrobial compounds. In addition, lichen-associated bacteria have been described to harbor ecological functions crucial for the evolution of the lichen holobiont. Nevertheless, the ecological features of lichen-associated microbes are still underestimated. To explore the untapped ecological diversity of lichen-associated bacteria, we adopted a novel culturomic approach on the crustose lichen Rhizocarpon geographicum. We sampled R. geographicum in French habitats exposed to oil spills, and we combined nine culturing methods with 16S rRNA sequencing to capture the greatest bacterial diversity. A deep functional analysis of the lichen-associated bacterial collection showed the presence of a set of bacterial strains resistant to a wide range of antibiotics and displaying tolerance to Persistent Organic Pollutants (POPs). Our study is a starting point to explore the ecological features of the lichen microbiota.

Roseburia intestinalis Modulates PYY Expression in a New a Multicellular Model including Enteroendocrine Cells.

The gut microbiota contributes to human health and disease; however, the mechanisms by which commensal bacteria interact with the host are still unclear. To date, a number of in vitro systems have been designed to investigate the host-microbe interactions. In most of the intestinal models, the enteroendocrine cells, considered as a potential link between gut bacteria and several human diseases, were missing. In the present study, we have generated a new model by adding enteroendocrine cells (ECC) of L-type (NCI-H716) to the one that we have previously described including enterocytes, mucus, and M cells. After 21 days of culture with the other cells, enteroendocrine-differentiated NCI-H716 cells showed neuropods at their basolateral side and expressed their specific genes encoding proglucagon (GCG) and chromogranin A (CHGA). We showed that this model could be stimulated by commensal bacteria playing a key role in health, Roseburia intestinalis and Bacteroides fragilis, but also by a pathogenic strain such as Salmonella Heidelberg. Moreover, using cell-free supernatants of B. fragilis and R. intestinalis, we have shown that R. intestinalis supernatant induced a significant increase in IL-8 and PYY but not in GCG gene expression, while B. fragilis had no impact. Our data indicated that R. intestinalis produced short chain fatty acids (SCFAs) such as butyrate whereas B. fragilis produced more propionate. However, these SCFAs were probably not the only metabolites implicated in PYY expression since butyrate alone had no effect. In conclusion, our new quadricellular model of gut epithelium could be an effective tool to highlight potential beneficial effects of bacteria or their metabolites, in order to develop new classes of probiotics.