New insights into the Van Krevelen diagram: Automated molecular formula determination from HRMS for a large chemical profiling of lichen extracts.

INTRODUCTION: In recent years, LC-MS has become the golden standard for metabolomic studies. Indeed, LC is relatively easy to couple with the soft electrospray ionization. As a consequence, many tools have been developed for the structural annotation of tandem mass spectra. However, it is sometimes difficult to do data-dependent acquisition (DDA), especially when developing new methods that stray from the classical LC-MS workflow. OBJECTIVE: An old tool from petroleomics that has recently gained popularity in metabolomics, the Van Krevelen diagram, is adapted for an overview of the molecular diversity profile in lichens through high-resolution mass spectrometry (HRMS). METHODS: A new method is benchmarked against the state-of-the-art classification tool ClassyFire using a database containing most known lichen metabolites (n ≈ 2,000). Four lichens known for their contrasted chemical composition were selected, and extractions with apolar, aprotic polar, and protic polar solvents were performed to cover a wide range of polarities. Extracts were analyzed with direct infusion electrospray ionization mass spectrometry (DI-ESI-MS) and atmospheric solids analysis probe mass spectrometry (ASAP-MS) techniques to be compared with the chemical composition described in the literature. RESULTS: The most common lichen metabolites were efficiently classified, with more than 90% of the molecules in some classes being matched with ClassyFire. Results from this method are consistent with the various extraction protocols in the present case study. CONCLUSION: This approach is a rapid and efficient tool to gain structural insight regarding lichen metabolites analyzed by HRMS without relying on DDA by LC-MS/MS analysis. It may notably be of use during the development phase of novel MS-based metabolomic approaches.

ASAP-MS and DART-MS as ancillary tools for direct analysis of the lichen metabolome.

INTRODUCTION: Lichens contain unique metabolites that most often need to be characterized from a limited amount of material. While thin layer chromatography is still the preferred analysis method for most lichenologists, liquid chromatography gives a deeper insight in the lichen metabolome, but an extractive step is needed before any analysis. Therefore, ambient ionization mass spectrometry (MS) analysis of lichen samples using Atmospheric Solid Analysis Probe (ASAP) and Direct Acquisition in Real Time (DART) techniques is evaluated. OBJECTIVE: We looked for a faster method to screen the metabolome by disrupting the classical workflow of analysis. METHODS: Four lichens selected for their metabolic diversity were analyzed with MS; namely Evernia prunastri, Lichina pygmaea, Parmelia saxatilis, and Roccella fuciformis. ASAP and DART analyses were compared against the reference electrospray ionization with a bioinformatic process including Van Krevelen diagrams as well as the multivariate comparison of the ionization methods in positive and negative modes. RESULTS: Metabolite profiles obtained from DART and ASAP analyses of lichen samples are consistent with classical analyses of lichen extracts. Through an easy and rapid experiment and without any extraction solvent, a large and informative profile of lichen metabolites is obtained when using complementary ionization modes of these high resolution mass spectrometry methods. CONCLUSION: ASAP-MS and DART-MS are two ancillary methods that provide a comprehensive evaluation of the lichen metabolome.

Inhibitory Effects of Secondary Metabolites from the Lichen Stereocaulon evolutum on Protein Tyrosine Phosphatase 1B.

Protein tyrosine phosphatase 1B plays a significant role in type 2 diabetes mellitus and other diseases and is therefore considered a new drug target. Within this study, an acetone extract from the lichen Stereocaulon evolutum was identified to possess strong protein tyrosine phosphatase 1B inhibition in a cell-free assay (IC50 of 11.8 µg/mL). Fractionation of this bioactive extract led to the isolation of seven known molecules belonging to the depsidones and the related diphenylethers and one new natural product, i.e., 3-butyl-3,7-dihydroxy-5-methoxy-1(3H)-isobenzofurane. The isolated compounds were evaluated for their inhibition of protein tyrosine phosphatase 1B. Two depsidones, lobaric acid and norlobaric acid, and the diphenylether anhydrosakisacaulon A potently inhibited protein tyrosine phosphatase 1B with IC50 values of 12.9, 15.1, and 16.1 µM, respectively, which is in the range of the protein tyrosine phosphatase 1B inhibitory activity of the positive control ursolic acid (IC50 of 14.4 µM). Molecular simulations performed on the eight compounds showed that i) a contact between the molecule and the four main regions of the protein is required for inhibitory activity, ii) the relative rigidity of the depsidones lobaric acid and norlobaric acid and the reactivity related to hydrogen bond donors or acceptors, which interact with protein tyrosine phosphatase 1B key amino acids, are involved in the bioactivity on protein tyrosine phosphatase 1B, iii) the cycle opening observed for diphenylethers decreased the inhibition, except for anhydrosakisacaulon A where its double bond on C-8 offsets this loss of activity, iv) the function present at C-8 is a determinant for the inhibitory effect on protein tyrosine phosphatase 1B, and v) the more hydrogen bonds with Arg221 there are, the more anchorage is favored.

Mass Spectrometry Imaging of Specialized Metabolites for Predicting Lichen Fitness and Snail Foraging.

Lichens are slow-growing organisms supposed to synthetize specialized metabolites to protect themselves against diverse grazers. As predicted by the optimal defense theory (ODT), lichens are expected to invest specialized metabolites in higher levels in reproductive tissues compared to thallus. We investigated whether Laser Desorption Ionization coupled to Mass Spectrometry Imaging (LDI-MSI) could be a relevant tool for chemical ecology issues such as ODT. In the present study, this method was applied to cross-sections of thalli and reproductive tissues of the lichen Pseudocyphellaria crocata. Spatial mapping revealed phenolic families of metabolites. A quantification of these metabolites was carried out in addition to spatial imaging. By this method, accumulation of specialized metabolites was observed in both reproductive parts (apothecia and soralia) of P. crocata, but their nature depended on the lichen organs: apothecia concentrated norstictic acid, tenuiorin, and pulvinic acid derivatives, whereas soralia mainly contained tenuiorin and pulvinic acid. Stictic acid, tenuiorin and calycin, tested in no-choices feeding experiments, were deterrent for N. hookeri while entire thalli were consumed by the snail. To improve better knowledge in relationships between grazed and grazing organisms, LDI-MSI appears to be a complementary tool in ecological studies.

Overcoming deterrent metabolites by gaining essential nutrients: A lichen/snail case study.

Specialised metabolites in lichens are generally considered repellent compounds by consumers. Nevertheless, if the only food available is lichens rich in specialised metabolites, lichenophages must implement strategies to overcome the toxicity of these metabolites. Thus, the balance between phagostimulant nutrients and deterrent metabolites could play a key role in feeding preferences. To further understand lichen-gastropod interactions, we studied the feeding behaviour and consumption in Notodiscus hookeri, the land snail native to sub-Antarctic islands. The lichen Usnea taylorii was used because of its simple chemistry, its richness in usnic acid (specialised metabolite) and arabitol (primary metabolite) and its presence in snail habitats. Choice tests in arenas with intact lichens versus acetone-rinsed lichens were carried out to study the influence of specialised metabolites on snail behaviour and feeding preference. Simultaneously, usnic acid and arabitol were quantified and located within the lichen thallus using HPLC-DAD-MS and in situ imaging by mass spectrometry to assess whether their spatial distribution explained preferential snail grazing. No-choice feeding experiments, with the pure metabolites embedded in an artificial diet, defined a gradual gustatory response, from strong repellence (usnic acid) to high appetence (D-arabitol). This case study demonstrates that the nutritional activity of N. hookeri is governed by the chemical quality of the food and primarily by nutrient availability (arabitol), despite the presence of deterrent metabolite (usnic acid).

Intrathalline Metabolite Profiles in the Lichen Argopsis friesiana Shape Gastropod Grazing Patterns.

Lichen-gastropod interactions generally focus on the potential deterrent or toxic role of secondary metabolites. To better understand lichen-gastropod interactions, a controlled feeding experiment was designed to identify the parts of the lichen Argopsis friesiana consumed by the Subantarctic land snail Notodiscus hookeri. Besides profiling secondary metabolites in various lichen parts (apothecia, cephalodia, phyllocladia and fungal axis of the pseudopodetium), we investigated potentially beneficial resources that snails can utilize from the lichen (carbohydrates, amino acids, fatty acids, polysaccharides and total nitrogen). Notodiscus hookeri preferred cephalodia and algal layers, which had high contents of carbohydrates, nitrogen, or both. Apothecia were avoided, perhaps due to their low contents of sugars and polyols. Although pseudopodetia were characterized by high content of arabitol, they were also rich in medullary secondary compounds, which may explain why they were not consumed. Thus, the balance between nutrients (particularly nitrogen and polyols) and secondary metabolites appears to play a key role in the feeding preferences of this snail.

The Hypotriglyceridemic Effect of Sciadonic Acid is Mediated by the Inhibition of Δ9-Desaturase Expression and Activity.

SCOPE: Sciadonic acid (Scia; 20:3Δ5,11,14) is a distinctive fatty acid (FA) with a polymethylene-interrupted double bond at C5. It is specifically found in seeds from gymnosperms such as pine nuts. Published papers describe a decrease in liver and plasma triacylglycerols in rats fed with this nutriment. The present study seeks to identify the action mechanism of Scia on triacylglycerol synthesis. In this way, its nutritional effect on FA metabolism involving the Stearoyl-CoA Desaturase 1 (SCD1) is investigated. METHODS AND RESULTS: Scia is discerned in trace amount in various tissues of rats and in human serum. It is produced by Δ5-desaturation of 20:2n-6 in human transfected SH-SY5Y cell lines and also in rat hepatocytes. When Scia is incubated with cultured hepatocytes as a nutrient, the cellular FA profile is modified. In particular, the proportion of the monoenes (18:1n-9, 18:1n-7, 16:1n-7) are all decreased, correlating to the reduction of triacylglycerol amounts. This effect is mediated by the inhibition of SCD1 expression. Furthermore, Scia, as well as 20:3n-6 and 20:3n-9 but not 20:3n-3, strongly inhibit the SCD1 activity measured on liver microsomes. CONCLUSION: Overall, this study shows that Scia, despite its unusual structure, contributes to the FA metabolism and reduced triacylglycerol release by inhibiting SCD1 activity.

Specialized Metabolites of the Lichen Vulpicida pinastri Act as Photoprotective Agents.

The extreme resiliency of lichens to UV radiations makes them an interesting model to find new photoprotective agents acting as UV-blockers and antioxidant. In this research, using a new in vitro method designed to overcome the shortage of material associated to many studies dealing with natural products, we show that the three major compounds isolated from the lichen Vulpicida pinastri, vulpinic acid, pinastric acid and usnic acid, were UV blocker agents. Antioxidant assays evidenced superoxide anion scavenging activity. Combination of the most promising compounds against UVB and UVB radiations, usnic acid, vulpinic acid and pinastric acid, increased the photoprotective activity. At the same time, they were found not cytotoxic on keratinocyte cell lines and photostable in the UVA and UVB ranges. Thus, lichens represent an attractive source to find good candidate ingredients as photoprotective agents. Additionally, the uncommon scalemic usnic acid mixture in this Vulpicida species was proven through electronic circular dichroism calculation.

Spatial mapping of lichen specialized metabolites using LDI-MSI: chemical ecology issues for Ophioparma ventosa.

Imaging mass spectrometry techniques have become a powerful strategy to assess the spatial distribution of metabolites in biological systems. Based on auto-ionisability of lichen metabolites using LDI-MS, we herein image the distribution of major secondary metabolites (specialized metabolites) from the lichen Ophioparma ventosa by LDI-MSI (Mass Spectrometry Imaging). Such technologies offer tremendous opportunities to discuss the role of natural products through spatial mapping, their distribution patterns being consistent with previous chemical ecology reports. A special attention was dedicated to miriquidic acid, an unexpected molecule we first reported in Ophioparma ventosa. The analytical strategy presented herein offers new perspectives to access the sharp distribution of lichen metabolites from regular razor blade-sectioned slices.

Integrating targeted gene expression and a skin model system to identify functional inhibitors of the UV activated p38 MAP kinase.

The stress-activated p38α MAP Kinase is an integral and critical component of the UV-induced inflammatory response. Despite the advances in recent years in the development of p38 kinase inhibitors, validation of these compounds in the diseased models remains limited. Based on the pharmacological profile of p38α inhibitor lead compound, SB203580, we synthesized a series of pyrrole-derivatives. Using UV-irradiated human skin punch-biopsies and cell cultures, we identified and validated the inhibitory activity of the derivatives by quantitatively measuring their effect on the expression of p38α target genes using real-time PCR. This approach not only identified pyrrole-2 as a unique derivative of this series that specifically inhibited the UV-activated p38α kinase, but also documented the skin permeation, bioavailability and reversible properties of this derivative in a 3D structure. The successful skin permeation of pyrrole-2 and its impact on AREG, COX-2 and MMP-9 gene expression demonstrates its potential use in modulating inflammatory processes in the skin. This study underscored the importance of using adapted biological models to identify accurate bioactive compounds.

Depsides: lichen metabolites active against hepatitis C virus.

A thorough phytochemical study of Stereocaulon evolutum was conducted, for the isolation of structurally related atranorin derivatives. Indeed, pilot experiments suggested that atranorin (1), the main metabolite of this lichen, would interfere with the lifecycle of hepatitis C virus (HCV). Eight compounds, including one reported for the first time (2), were isolated and characterized. Two analogs (5, 6) were also synthesized, to enlarge the panel of atranorin-related structures. Most of these compounds were active against HCV, with a half-maximal inhibitory concentration of about 10 to 70 µM, with depsides more potent than monoaromatic phenols. The most effective inhibitors (1, 5 and 6) were then added at different steps of the HCV lifecycle. Interestingly, atranorin (1), bearing an aldehyde function at C-3, inhibited only viral entry, whereas the synthetic compounds 5 and 6, bearing a hydroxymethyl and a methyl function, respectively, at C-3 interfered with viral replication.

Lichenic extracts and metabolites as UV filters.

Three lichen extracts and ten lichenic compounds have been screened for their photoprotective activities. The determination of their Sun Protection Factor (SPF) and Protection Factor-UVA (PF-UVA) values was done in vitro. Among them, a Lasallia pustulata extract and gyrophoric acid exhibited SPF values over 5, which is better than Homosalate (SPF≈4). Their photoprotective properties are only slightly modified after a 2-hours period of irradiation. Salazinic acid and L. pustulata presented characteristics of a UVA booster like the butyl-methoxydibenzoylmethane (Avobenzone) (PF-UVA≈2 vs. 2.8 for Avobenzone). Salazinic acid was a better anion superoxide scavenger than ascorbic acid and none of them exhibited a photosensitizing cytotoxicity by exposing them on HaCaT cells to UVA radiations (photo-irritancy factor PIF<5).

Antifungal activity of 10 Guadeloupean plants.

Screening of the antifungal activities of ten Guadeloupean plants was undertaken to find new extracts and formulations against superficial mycoses such as onychomycosis, athlete's foot, Pityriasis versicolor, as well as the deep fungal infection Pneumocystis pneumonia. For the first time, the CMI of these plant extracts [cyclohexane, ethanol and ethanol/water (1:1, v/v)] was determined against five dermatophytes, five Candida species, Scytalidium dimidiatum, a Malassezia sp. strain and Pneumocystis carinii. Cytotoxicity tests of the most active extracts were also performed on an HaCat keratinocyte cell line. Results suggest that the extracts of Bursera simaruba, Cedrela odorata, Enterolobium cyclocarpum and Pluchea carolinensis have interesting activities and could be good candidates for developing antifungal formulations.

Lobarin from the Sumatran lichen, Stereocaulon halei.

The diphenyl ether, lobarin (1) (syn. lobariol carboxylic acid) related to lobaric acid was isolated for the first time as a natural product along with five known compounds from Stereocaulon halei, a fruticose lichen collected in Indonesia. The structure of lobarin was elucidated by spectroscopic data analysis and its most stable conformers were determined by molecular mechanic dynamic calculations. A marked superoxide anion scavenging was found for compound 1 while no cytotoxicity on the B16 murine melanoma and HaCaT human keratinocyte cell lines was observed.

Stictic acid derivatives from the lichen Usnea articulata and their antioxidant activities.

Two new beta-orcinol depsidones, 1 and 2, together with 13 known compounds were isolated from the lichen Usnea articulata. The structures of 1 and 2 were elucidated by spectroscopic analyses and those of known compounds by comparison of their spectroscopic data with literature values or by direct comparison with authentic standards. Compounds 1, 2, and 5 exhibited moderate antiradical activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The depsidones 4 and 5 showed better superoxide anion scavenging activity (IC50 = 566 and 580 microM, respectively) than quercetin (IC50 = 754 microM).

Flavonols from Scurrula ferruginea Danser (Loranthaceae).

Three natural flavonols compounds have been isolated from the ethyl acetate fraction of Scurrula ferruginea Danser (Loranthaceae). Besides quercetin and quercitrin, an unusual flavonol glycoside 4"-O-acetylquercitrin was isolated. Structures were determined using spectroscopic methods including UV, NMR and HRMS-EI. The incidence of 4"-O-acetylquercitrin, not previously reported in the Loranthaceae, is discussed. Cytotoxic evaluation on four human cancer cell lines showed quercetin to be the most active with IC50 of 35 microM on U251 (human glioblastoma cells).