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. 2026 Sep 17;11(38):57213–57221. doi: 10.1021/acsomega.6c05106

Lichens as a Source of Nematocidal Compounds: Identification of Sphaerophorin and Divaricatic Acid Active against Caenorhabditis elegans

Richard Frenák †,*, Zuzana Vaneková ‡, Simone Lager ‡, Viktória Medvecová §, Martin Kello §, Judith M Rollinger ‡, Michal Goga †,∥
PMCID: PMC13628625  PMID: 42824575

Abstract

Increasing helminth drug resistance necessitates novel anthelmintic agents. Lichens, historically used to treat parasitic infections, represent a promising natural resource. This study evaluated eight lichen extracts and 17 metabolites against the model organism Caenorhabditis elegans (wild-type strain N2). Extracts of Hypogymnia physodes, Nephroma arcticum, and Stereocaulon grande showed significant efficacy, while Sphaerophorus fragilis and Ophioparma ventosa caused 100% nematode mortality on day 11 (at 25 and 100 μg/mL, respectively). Chemical workup of S. fragilis yielded squamatic acid, sphaerophorin, and its monocyclic degradation products. Among the tested compounds, the depside sphaerophorin exhibited the highest nematocidal activity (LC50 = 22.07 μM), followed by divaricatic acid (LC50 = 77.27 μM). Physodic acid (LC50 > 200 μM) and evernic acid showed moderate effects. Sphaerophorin and divaricatic acid demonstrated moderate to low selectivity, generally exhibiting lower cytotoxicity toward human CCD-18Co cells (CC50 values of 88.5 μM and 91.7 μM, respectively) than toward nematodes. Given a panel of 17 structurally related compounds, we conducted a structure–activity relationship (SAR) analysis to identify molecular patterns associated with observed nematocidal activity. The analysis suggested the importance of the intact depside structure together with the presence of aliphatic chains and a carboxylic group, as isolated monocyclic fragments lacked antinematode efficacy. This first report on the nematocidal activity of H. physodes, N. arcticum, S. grande, and S. fragilis extracts, alongside evernic acid, physodic acid, and sphaerophorin, against the model organism C. elegans identifies them as promising scaffolds that warrant further investigation against parasitic species.

graphic file with name ao6c05106_0008.webp

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Introduction

Parasitic helminths represent one of the most successful biological strategies on Earth, adapting to virtually every type of animal host. Infections caused by these organisms, collectively known as helminthiases, pose a persistent threat to global health and agricultural economics. Approximately one-third of the human population is infected with helminth parasites, including widespread soil-transmitted nematodes such as Ascaris lumbricoides and Trichuris trichiura, as well as vector-borne diseases like lymphatic filariasis. − Beyond human morbidity, plant-parasitic nematodes, including root-knot nematodes (Meloidogyne spp.), cause estimated annual losses of USD 125 billion to global agriculture.

Currently, control of these infections relies heavily on a limited arsenal of synthetic anthelmintics. However, the efficacy of these treatments is increasingly compromised. Intensive use of these compounds has accelerated the selection of drug-resistant parasite strains, a phenomenon now widespread in livestock and emerging in human populations. Furthermore, the discovery of new drug classes has lagged, creating a critical need for novel therapeutic agents with distinct mechanisms of action that can overcome existing resistance. In this context, natural products have historically served as a rich source of bioactive scaffolds. While higher plants have been extensively screened, lichenssymbiotic associations between fungi and photoautotrophsremain an underexplored reservoir of potential antiparasitic compounds.

Lichens have been an integral part of Traditional Medicine in the treatment of parasitic infections for millennia. Their use is documented across diverse cultures, from the “chharila” of Ayurveda in India to the medicinal practices of the Cherokee in North America and Traditional Chinese Medicine. , In early modern Europe, infusions of Peltigera aphthosa and powdered Pertusaria pertusa were commonly applied for their vermicidal properties. , These traditional applications suggest that lichens possess inherent anthelmintic properties, likely attributable to their unique secondary metabolites.

Chemotaxonomically, lichens are recognized by their distinctive secondary metabolites, including depsides, depsidones, dibenzofurans, and phenolic acids, which are synthesized primarily via the acetyl-polymalonyl pathway. , Despite the broad pharmacological potential of these compoundsranging from antibiotic to anticancer activitiesscientific verification of their anthelmintic effects remains limited compared with other biological activities. , Early studies demonstrated that extracts from Evernia prunastri and some of its phenolic constituents were highly lethal to Toxocara canis larvae. More recently, usnic acid, a major dibenzofuran derivative, and its potassium salt showed potent activity against Schistosoma mansoni, causing tegumental damage and mortality in both larval and adult stages. , Barbatic and divaricatic acids from the depside group also proved effective against the same parasitic model organism. , Similarly, lecanoric acid from Parmelia cetrata has shown efficacy against the nematode model Caenorhabditis elegans.

Despite these promising findings, the mechanism of action of lichen metabolites against helminths remains unclear. Current literature suggests that natural products may act through multitarget mechanisms, including neuromuscular disruption, interference with metabolic pathways, and damage to cellular membranes. However, research on lichens is limited to a small number of species and compounds. Bridging the gap between traditional ethnobotanical knowledge and modern molecular pharmacology offers a promising avenue for discovering new anthelmintic candidates.

This study aims to evaluate the nematocidal potential of eight selected lichen extracts from Bryoria fuscescens, Sphaerophorus fragilis, Ophioparma ventosa, Cetraria aculeata, Nephroma arcticum, Stereocaulon grande, and Lobaria pulmonaria, as well as 17 secondary metabolites. By isolating bioactive fractions and assessing their efficacy against C. elegans, a model organism, this research seeks to validate historical lichen applications and identify novel structural features to guide the development of anthelmintic compounds. Furthermore, given the large number of structurally similar compounds tested, a structure–activity relationship can be inferred.

Results and Discussion

Effect of Lichen Extracts on C. elegans Survival

Due to its ease of cultivation and high degree of genetic and physiological similarity to parasitic worms, the free-living nematode Caenorhabditis elegans represents a highly suitable model organism for screening the anthelmintic activity of natural products. − The search for new antiparasitics is crucial, as helminthiases cause significant harm to human health and agriculture, leading to economic instability and increased mortality.

In our initial screening, eight selected lichen acetone extracts were evaluated using a survival assay. After 11 days of exposure (Figure A), a significant decrease in nematode survival relative to the control was observed for the extracts of H. physodes (100 μg/mL), N. arcticum (100 μg/mL), and O. ventosa (25 μg/mL). Complete mortality (100%) was recorded for O. ventosa (100 μg/mL) and S. fragilis (at both 100 and 25 μg/mL). Extracts that did not show immediate nematocidal effects were monitored up to day 14 (Figure B). By this time, a further statistically significant decrease in survival was observed for the S. grande extract (100 μg/mL), and complete mortality was recorded for N. arcticum.

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Survival assay of C. elegans treated with acetone extracts of selected lichens after 11 (A) and 14 (B) days at two concentrations (25 and 100 μg/mL). The experiments were performed in parallel in three triplicates (n = 9). Data are presented as mean ± standard deviation (SD). Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 compared to the control (0.7% DMSO).

The high activity of the S. fragilis and O. ventosa extracts can be largely attributed to their major metabolites. However, the exact driver of activity in O. ventosa requires careful interpretation. While divaricatic acid (DVA) is a major component, the presence of usnic acid (UA)which is abundant in this species and has known effects against the parasite Schistosoma mansoni , must also be considered. Because pure UA was not tested against C. elegans in our study and its exact concentration in the extracts was not quantified, its contribution to the overall activity remains to be determined. Similarly, the delayed activity of the N. arcticum extract warrants further study. Although its typical metabolites (methyl gyrophorate, nephroarctin, phenarctin, zeorin) have not yet been investigated for anthelmintic properties, UA is also present in this species. The moderate effect of the H. physodes extract is likely related to its main constituent, physodic acid (PHY).

Identification and Screening of Lichen Secondary Metabolites on C. elegans

To identify the specific compounds responsible for the observed activities, the active acetone extract of S. fragilis (Figure S1) was fractionated by flash chromatography and Sephadex dextran gel purification. This approach enabled the isolation of four compounds, identified by NMR spectroscopy as the depsides squamatic acid (SQA) (Figure S3) and sphaerophorin (SPH) (Figure S5), along with two monocyclic fragments: everninic acid (EVEA) (Figure S2) and sphaerophorolcarboxylic acid (SPHCA) (Figure S4). We hypothesize that these fragments formed as artifacts via autolysis during Soxhlet extraction at elevated temperature, a phenomenon previously described for atranorin.

Consequently, a diverse set of 17 secondary metabolitesconsisting of depsides, depsidones, and phenolic acids (Table S5)was compiled from our isolated compounds and an internal collection. All compounds are known to occur in lichens and were identified by comparing their NMR data with published data. After 5 days of exposure, strong nematocidal activity was observed for DVA and SPH, with survival rates plummeting to 2% and 3%, respectively, compared with 88% in the DMSO control (Figure A). A statistically significant decrease in live nematodes was also observed for PHY (61%) and evernic acid (EA) (64%). A second reading on day 12 confirmed this trend: survival decreased to 6% for PHY and 33% for EA, while DVA and SPH exhibited 100% mortality (Figure B). A mild, statistically nonsignificant inhibitory effect was observed for 3-hydroxyphysodic acid (3OH), α-alectoronic acid (α-ALE), and SPHCA. None of the other metabolites tested showed a positive effect on survival compared with the control group, even after 14 days (data not shown).

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Survival assay of C. elegans treated with lichen secondary metabolites after 5 (A) and 12 (B) days at 100 μM concentration. The experiments were performed in parallel in three triplicates (n = 9). ATR – atranorin, Cl-ATR – chloroatranorin, PHY – physodic acid, 3OH – 3-hydroxyphysodic acid, PHDL – physodalic acid, EA – evernic acid, GA – gyrophoric acid, DVA – divaricatic acid, STA – stictic acid, NRSTA – norstictic acid, CRSTA – cryptostictic acid, α-ALE – α-alectoronic acid, EVEA – everninic acid, SPHCA – sphaerophorolcarboxylic acid, SPH – sphaerophorin. Data are presented as mean ± standard deviation (SD). Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 compared to the control (1% DMSO).

Concentration-Dependent Nematocidal Activity

Compounds with the most pronounced nematocidal activity were further evaluated across a range of concentrations. SPH exhibited the highest potency, showing inhibition at concentrations as low as 1 μM from day 7. Complete nematode mortality occurred at 100 and 50 μM after 5 days, and at 25 and 10 μM on day 12 (Figure A). DVA showed a slightly lower but still substantial effect; survival at 100 and 50 μM approached zero after 7 days, and at 25 and 10 μM by days 12 and 15, respectively (Figure B).

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Effect of (A) sphaerophorin and (B) divaricatic acid on the survival of C. elegans across a concentration range of 1, 10, 25, 50, and 100 μM. The proportion of live nematodes was recorded on days 3, 5, 7, 10, 12, and 14/15. The experiments were performed in parallel in three triplicates (n = 9). Data are presented as mean ± standard deviation (SD). Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 compared to the control (1% DMSO).

Physodic acid (PHY) showed mild activity at all tested concentrations as early as day 5. Complete mortality was achieved only at the highest concentrations (200 μM and 100 μM on days 12 and 15, respectively), but notable long-term effects were observed at lower doses. By the end of the experiment, survival rates in the 50 μM and 25 μM groups approached 9%. Importantly, an inhibitory effect was evident even at the lowest tested concentration of 10 μM, yielding a 34% survival rate compared with 59% in the control group (Figure ).

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Effect of physodic acid on the survival of C. elegans across a concentration range of 10, 25, 50, 100, and 200 μM. The proportion of live nematodes was recorded on days 3, 5, 7, 10, 12, and 15. The experiments were performed in parallel in three triplicates (n = 9). Data are presented as mean ± standard deviation (SD). Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 compared to the control (1% DMSO).

In previous experiments, we also tested different concentrations of levamisole as a positive control. We determined that 10 μM levamisole was best suited to reduce the nematodes’ lifespan and motility to about 10% after 3 days, 5% after 5 days, and <1% after 7 days. Using the same protocol, we did not include the levamisole control again in our testing of lichen samples. However, the protocol is well established and reliably demonstrates the distinct nematocidal effect of the tested lichen compounds.

To ensure comparability with commercial anthelmintics, LC50 values were calculated on day 3 of exposure. The LC50 was 22.07 μM for SPH, 77.27 μM for DVA, and >200 μM for PHY. Although these values are higher than those reported for commercial drugs evaluated on the same C. elegans strain after 16 h (paraherquamide A 5.1 μM), 24 h (avermectin 15.4 μM), and 48 h (levamisole 7.2 μM, ivermectin 1.3 μM, and albendazole 26.4 μM), our data were recorded after 72 h. Our LC50 values would likely be higher if determined earlier. Notably, this is the first report of nematocidal activity for both the S. fragilis extract and its major metabolite, sphaerophorin.

Structure–Activity Relationship (SAR) Analysis

With 17 structurally related compounds, we sought specific molecular patterns across the set of compounds assayed to identify correlations between structure and observed nematocidal activity. Analysis of the tested compounds revealed several key structure–activity correlations that build upon existing literature. In studies on Toxocara canis, Kondo et al. previously demonstrated that the activity of monoaromatic metabolites (resorcinol type) increases with the length of the aryl chain. Likewise, Dube et al. underscored the significance of long alkyl chains in anacardic acids, specifically in conjunction with a carboxyl group, for efficacy in the C. elegans model. Drugs reach nematodes either through ingestion or passive membrane transport. Therefore, the appropriate degree of lipophilicity is crucial for crossing the cuticle and cellular membranes to attain an effective concentration within the organism. However, lipophilicity alone does not entirely predict transport success. Since the presence of a carboxylic group is equally critical, it seems that amphiphilic character is advantageous for observed activity. Furthermore, Zorrilla et al. concluded that the o- and p- positions of −OH groups (the basic resorcinol structure) on the benzene ring correlate with anthelmintic activity toward Meloidogyne incognita.

Our results partially confirm these observations but provide an important clarification: resorcinol fragments alone, in the context of depsides (e.g., atranorin, chloroatranorin, gyrophoric acid), were ineffective against C. elegans. Activity was strictly limited to compounds combining a depside structure with aliphatic chains and a carboxylic group, thus having an amphiphilic nature (SPH and DVA) (Figure a,b). The only depsidone showing moderate activity was PHY, which also contains an aliphatic chain (Figure c). Interestingly, adding an −OH group to yield 3OH (Figure d) decreased activity. Although the resulting catechol fragment is a known antioxidant, this structural modification diminished nematocidal efficacy.

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Key features of (a) sphaerophorin, (b) divaricatic acid, (c) physodic acid and (d) 3-hydroxyphysodic acid molecules. Blue – carboxylic group; Green – aliphatic chain; Red – catechol fragment.

Perhaps the most fundamental finding of our SAR analysis is that the breakdown of the sphaerophorin molecule into its monoaromatic components (EVEA and SPHCA) results in a complete loss of nematocidal efficacy. This suggests that the intact depside scaffoldnot merely the presence of the aliphatic chain and/or carboxylic groupplays a critical role in maintaining nematocidal activity.

Cytotoxicity and Safety Profile

It is often suggested in the literature that the anthelmintic activity of polyphenols is nonspecific, driven by mechanisms such as hydrogen bonding or general antioxidant activity, and therefore correlates with toxicity toward host cells. To address this, we evaluated the cytotoxicity of our most potent compounds (SPH and DVA) using a resazurin assay on human colon fibroblasts (CCD-18Co)a highly relevant model, as the colon is a primary habitat for intestinal helminths.

The two compounds showed differing profiles regarding selective toxicity toward nematodes. SPH and DVA exhibited CC50 values in human fibroblasts of 88.5 μM and 91.7 μM, respectively (Table ), yielding selectivity indices of 4.21 (for SPH) and 1.18 (for DVA). In the present experimental setting, DVA does not prove to be a selective agent. Conversely, a favorable safety profile for DVA was previously reported for aquatic invertebrates (Artemia salina) and human mononuclear cells, ,,, indicating that more research is needed to characterize its selectivity fully.

1. LC50/CC50 Values (μM) and Selectivity Indices (SI = CC50/LC50) of Sphaerophorin and Divaricatic Acid Calculated from Concentration-Dependent Survival Assays on C. Elegans and Cytotoxicity Assay on CCD-18Co .

  LC50 (C. elegans) CC50 (CCD-18Co) Selectivity index (SI)
Sphaerophorin 22.07 (95% CI = 12.02–28.05) 88.5 (95% CI = 83.1–93.9) 4.21
Divaricatic acid 77.27 (95% CI = 69.93–86.52) 91.7 (95% CI = 81.2–102.3) 1.18
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a

Cells after 72 h (day 3).

Limitations of the Study

While this study successfully identifies the anthelmintic potential of sphaerophorin and divaricatic acid, certain limitations of the current experimental design must be acknowledged. Although C. elegans is widely accepted as an initial screening platform, results on nematocidal activity do not necessarily translate to true parasitic helminths, as free-living nematodes lack the specific host-evasion mechanisms and physiological adaptations of obligate parasites. Consequently, the compounds identified herein should be viewed as early-stage chemical scaffolds or initial hits rather than advanced therapeutic leads.

Notably, a concurrent positive control was not included in this initial screening phase. To evaluate the relative potency of our compounds, we have referenced literature values; however, we recognize that variations in laboratory environments and assay parameters can complicate direct comparisons with external data. To mitigate this and ensure the reliability of our findings, the assays in this study were conducted using a strictly standardized protocol identical to that used in our previously published work. Therefore, while the lack of a concurrent positive control is a limitation, the historical consistency of this assay within our laboratory provides a useful reference for contextualizing and estimating the expected range of the LC50 values reported herein.

Regarding SAR analysis, no mechanistic investigations were conducted. All findings are derived from straightforward observations of the molecular structures of the compounds examined and the presence or absence of specific chemical moieties. It is also pertinent to note that the observable trends are based on a limited set of tested compounds.

Finally, the preliminary safety profile of these compounds was evaluated using a single human cell line (CCD-18Co). While this approach provides a valuable preliminary indication of general cytotoxicity and enables calculation of an initial selectivity index, it does not fully capture potential tissue-specific toxicities. Future development will require counter-screening against a broader panel of mammalian cell lines, along with parasitic models, and concurrent reference drugs, to comprehensively evaluate the therapeutic potential and safety of these compounds.

Conclusion

This study investigated the anthelmintic potential of lichen extracts and their secondary metabolites, a class of natural products with well-documented biological activities but limited exploration in antiparasitic therapy. Primary screening of eight acetone extracts revealed significant nematocidal activity in extracts from Sphaerophorus fragilis and Ophioparma ventosa, both causing 100% mortality in the model organism Caenorhabditis elegans at concentrations of 25 and 100 μg/mL.

Subsequent analysis of 17 isolated metabolites identified sphaerophorin (SPH) and divaricatic acid (DVA) as the most potent compounds. Sphaerophorin exhibited the strongest effect (LC50 = 22.07 (95% CI = 12.02–28.05) μM), with inhibitory activity observed even at low concentrations (1 μM) following prolonged exposure. Divaricatic acid showed moderate activity, with an LC50 of 77.27 (95% CI = 69.93–86.52) μM. Although both compounds exhibited similar cytotoxicity toward human colon fibroblasts (CCD-18Co), with CC50 values of 88.5 μM (95% CI = 83.1–93.9) for SPH and 91.7 μM (95% CI = 81.2–102.3) for DVA, their selectivity indices differed. SPH demonstrated moderate selectivity (SI = 4.21), whereas DVA showed low selectivity (SI = 1.18), failing to show substantially lower cytotoxicity toward human cells compared with its lethality in C. elegans.

To identify the structural prerequisites underlying this antinematode effect, a phenotypic SAR analysis was conducted. The results, although based on a limited number of compounds, suggest that toxicity against C. elegans depends heavily on the integrity of the depside scaffold combined with an amphiphilic profilespecifically, long lipophilic aliphatic chains paired with a polar carboxylic acid group. Conversely, the breakdown of these molecules into isolated monoaromatic fragments resulted in a complete loss of efficacy in this model.

While the anthelmintic activity of divaricatic acid has been previously described against Schistosoma mansoni, this study presents the first report of nematocidal activity of S. fragilis extract and its main constituent, sphaerophorin. Although the tested compounds did not match the potency of commercial reference drugs (such as levamisole or ivermectin), they represent promising initial hits amid rising resistance to synthetic anthelmintics.

The identification of these metabolites advances understanding of the structure–activity relationships of lichen depsides within a free-living nematode model. Moreover, these findings provide a preliminary scientific context for their application in traditional medicine and underscore the need for subsequent testing in parasitic species to evaluate their potential uses in pharmacology and agriculture. Structural optimization through semi-synthesis, aimed at enhancing bioavailability, potency, and safety profile, alongside the investigation of synergistic effects with existing pharmaceuticals, constitutes additional promising avenues for future development.

Experimental Section

Collection of Material

Thalli of the lichen Bryoria fuscescens (Gyelnik) Brodo & D. Hawksw. were collected in Sweden, and thalli of the lichens Sphaerophorus fragilis (L.) Pers., Ophioparma ventosa (L.) Norman, Cetraria aculeata (Schreber) Fr., and Nephroma arcticum (L.) Torss. were collected in Norway in September 2024 by Michal Goga and Anastasiia Tomliak. S. fragilis, O. ventosa, B. fuscescens, C. aculeata, and N. arcticum were identified by Dr. Goga and Prof. Bačkor as lichen specimens numbered KO38946, KO38944, KO38947, KO38948, and KO38943, respectively. The lichen Stereocaulon grande (H. Magn.) H. Magn. was collected by Michal Goga and Richard Frenák in Finland during September 2022. S. grande was determined by Dr. Goga to be a lichen specimen with the number KO38945. Thalli of Lobaria pulmonaria (L.) Hoffm. were sent to the Department of Plant Biology at UPJŠ through collaboration with the University of Pisa (Dr. Luca Paoli) in 2024. The local species Hypogymnia physodes (L.) Nyl. was collected in November 2023 from the bark of fallen trunks in the Volovské vrchy region (Kojšovská hol’a). Dr. Goga identified H. physodes as a lichen specimen numbered KO38942. All specimens are deposited in the herbarium of P.J. Šafárik in Košice, Slovakia.

Preparation of Extracts Used for Caenorhabditis elegans Testing

Collected thalli were mechanically cleaned of other plant material residues. For S. fragilis, the thalli were thoroughly washed with water to remove soil. Subsequently, the thalli were left to dry on filter paper at laboratory temperature. The material was macerated in acetone (Centralchem, min. 99.5%) for 6 h at a 1:10 (w/v) ratio (1 g of material per 10 mL of solvent). The supernatant was filtered through filter paper and evaporated using a rotary vacuum evaporator (Heidolph, Schwabach, Germany) under reduced pressure at 34 °C. Dry extracts were stored at 4 °C until testing.

Isolation of Lichen Secondary Metabolites

Procedures for isolating certain secondary metabolites were previously described by Elečko et al. The tested substances were obtained from the internal collection of standards of the Department of Plant Biology at UPJŠ in Košice. Secondary metabolites of S. fragilis were isolated using the infrastructure of the Department of Pharmacognosy at the Faculty of Life Sciences, University of Vienna. The crude extract was obtained by Soxhlet extraction with acetone for 8 h, yielding 1.3% of the lichen dry mass. Isolation steps included separation on normal- and reversed-phase columns via flash chromatography, followed by purification of the isolates using Sephadex LH-20 dextran gel. Chromatographic conditions for isolating secondary metabolites, together with a complete list of tested secondary metabolites, including their molecular weights and structural and molecular formulas (Table S5), are provided in the Supporting Information. All evaluated compounds were obtained with >95% purity, except squamatic acid, which exhibited a purity of 94.67% (Table S6, Figure S6).

UPLC Analysis of the Sphaerophorus fragilis Acetone Extract

A Waters Acquity UPLC instrument consisting of a sample manager, a quaternary solvent manager (QSM), a column manager, and a photodiode array (PDA) and evaporative light-scattering (ELSD) detector was used. The operating software was Empower 3. Stationary phase: Acquity BEH C18, 2.1 × 100, 1.7 μm; flow rate 0.3 mL/min. Mobile phase: Awater +0.1% formic acid, Bacetonitrile + 0.1% formic acid. Gradient program: 0 min 90/10% (A/B), 1 min 90/10% (A/B), 3 min 50/50% (A/B), 18 min 2/98% (A/B), 18.5 min 90/10% (A/B), 20 min 90/10% (A/B).

NMR Analysis of Secondary Metabolites

Isolates were dissolved in acetone-d6 at 3–5 mg/mL, centrifuged, and transferred to labeled NMR tubes. NMR experiments were conducted on a Bruker Advance 500 NMR spectrometer (UltraShield) equipped with a TCI Prodigy CryoProbe (5 mm, triple-resonance inverse-detection probe head). A full set of 1D and 2D NMR spectra was recorded: 1H (128 scans), 13C APT (4096 scans), HSQC (16 scans), HMBC (2 scans), COSY (4 scans), and NOESY (8 scans). NMR spectra were analyzed using MestReNova and evaluated manually.

Chemical shift values were checked against nmrdb.org and compared with experimental values from Huneck and Yoshimura.

Experiments with Caenorhabditis elegans

The wild-type C. elegans strain N2, var. Bristol, and Escherichia coli OP50 used in experimental survival assays were obtained from the Caenorhabditis Genetics Center at the University of Minnesota. Media and NGM (Nematode Growth Medium) agar plates were prepared according to Stiernagle. , E. coli OP50 and C. elegans cultures were maintained according to the protocol listed in Zwirchmayr et al. E. coli OP50 was cultured in LB medium for 8 h at 37 °C, then harvested by centrifugation, washed twice with double-distilled water, and air-dried. The bacterial pellet was resuspended in S-complete medium to 100 mg/mL and stored at 4 °C until use. The wild-type C. elegans strain N2, var. Bristol was maintained on NGM agar plates seeded with OP50 at 16 °C. Worms were monitored regularly and transferred weekly to fresh OP50-inoculated plates.

For testing purposes, worms were transferred to fresh NGM plates 3 days prior to synchronization. During the experiment, 96-well plates were stored at 25 °C. Survival assays were performed according to the method described in Redl et al. with slight modifications. For culture synchronization purposes, N2 C. elegans individuals were collected from NGM plates and suspended in ddH2O. The worms were exposed to an alkaline hypochlorite solution (bleaching solution) for 5–10 min to lyse the cuticles and release the eggs. Lysis was stopped by adding M9 buffer. Eggs were harvested by centrifugation at 2500× g, washed twice with M9 buffer and once with S-complete medium. Eggs were incubated in S-complete at room temperature on a shaker for 48 h until larvae hatched.

Survival Assays

8–18 L1-stage larvae were dispensed into the inner wells of three 96-well plates; the outer wells were filled with ddH2O and served as a diffusion barrier. Worms were fed fresh E. coliE. coli OP50 (5.6 mg/mL) and incubated at 25 °C. After 24 h, they were sterilized with 5-fluorodeoxyuridine (final concentration 0.12 mM; Sigma-Aldrich). The following day, young adults were treated with test samples and a solvent control (0.7% or 1% DMSO). Survival rates were calculated as the percentage of live worms relative to the initial number per well. Each experimental condition was tested in triplicate (three replicate wells per plate) and averaged. Final data are expressed as the mean ± standard deviation (SD) across three independent, parallel experiments. On the fourth day of adulthood, individuals were fed an additional 5 μL of OP50 solution (100 mg/mL) to prevent starvation. Twice a week, the plates were left open for a minute to oxygenate the worms’ environment. The specific test determined the evaluation day for worm survival, and assays generally lasted 14–15 days. Individuals were considered dead if they did not respond to mechanical stimulation or light exposure.

CCD-18Co Cell Culture

The cytotoxic effects of nematocidal secondary metabolites were evaluated using the normal human colon fibroblast cell line CCD-18Co (ATCC, Manassas, VA, USA). The CCD-18Co cells were cultured in DMEM (Biosera, Kansas City, MO, USA) supplemented with 10% FBS (fetal bovine serum; Gibco, Thermo Scientific, Rockford, IL, USA) and an antibiotic/antimycotic solution (Merck, Darmstadt, Germany). During the experiment, cells were maintained in an incubator at 37 °C in a humidified atmosphere containing 5% CO2.

Resazurin Cytotoxicity Assay

CCD-18Co cells were seeded into 96-well plates at 5 × 106 cells/well and cultured for 24 h under standard conditions. The isolated secondary metabolites (10, 50, and 100 μM) were then added to the cells in triplicate. Final concentrations of the tested compounds were prepared in complete medium with DMSO (vehicle, max. 0.2% v/v). After the experimental period (72 h), resazurin solution (Merck, Darmstadt, Germany) was added at 10 μL/well to achieve a final concentration of 40 μM. The metabolic activity of the cells following treatment with the tested lichen compounds was determined after at least 1 h of incubation by measuring fluorescence with a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA) using 560 nm excitation and 590 nm emission filters. The results were averaged and normalized to the control (100% fluorescence).

Statistical Analysis

Assay data were visualized as bar or XY graphs using GraphPad Prism 8.0.1 (GraphPad Software, La Jolla, CA, USA). For survival assays monitored over multiple days, each time point was treated as an independent dataset. A one-way ANOVA followed by Dunnett’s post hoc test was performed separately for each day to compare treatment concentrations with the vehicle control at that discrete end point. Differences were considered significant at p ≤ 0.05. Using day 3 data, relative predictive LC50 values were calculated using GraphPad Prism 8.0.1 linear regression analysis. The corresponding 95% confidence intervals (95% CI) for the mean LC50 values were calculated and are reported.

From the resulting cytotoxicity data, relative predictive CC50 values were calculated using the nonlinear regression function in GraphPad Prism 9.0.2 (GraphPad Software, La Jolla, CA, USA). The corresponding 95% confidence intervals (95% CI) for the mean CC50 values were computed and reported.

Supplementary Material

ao6c05106_si_001.pdf (566.4KB, pdf)

Acknowledgments

We thank Agnieszka Kowalska, Ulrike Grienke, Andreas Wasilewicz, Johannes Gafriller, Martina Redl, Eva Waltenberger, and Sigrid Adelsberger (all University of Vienna) for their valuable technical assistance and support.

The data presented in this study can be provided by the authors upon reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c05106.

  • Figure S1. UPLC chromatogram of the Soxhlet acetone extract of S. fragilis using ELSD detection; Table S1. 1H- and 13C-NMR data (δ, ppm) of everninic acid in acetone-d6; Figure S2. Chemical structure of everninic acid, along with the HMBC (blue) and NOESY (green) correlations; Table S2. 1H- and 13C-NMR data (δ, ppm) of squamatic acid in acetone-d6; Figure S3. Chemical structure of squamatic acid, along with the HMBC (blue) and NOESY (green) correlations; Table S3. 1H- and 13C-NMR data (δ, ppm) of sphaerophorolcarboxylic acid in acetone-d6; Figure S4. Chemical structure of sphaerophorolcarboxylic acid, along with the HMBC (blue), COSY (purple), and NOESY (green) correlations; Table S4. 1 H- and 13C-NMR data (δ, ppm) of sphaerophorin in acetone-d6, Figure S5. Chemical structure of sphaerophorin, along with the HMBC (blue), COSY (purple), and NOESY (green) correlations; Table S5. List of tested secondary metabolites with their designations, molecular weights, and molecular and structural formulas; Table S6. Purity data from UPLC/ELSD evaluation; Figure S6. UPLC/ELSD chromatograms for all lichen secondary metabolites. Values correspond to the % of the total peak area for each sample (PDF)

Conceptualization, J.M.R., R.F., and M.G.; Formal analysis, R.F.; Funding acquisition, M.G., J.M.R., R.F., and M.K.; Investigation, R.F., Z.V., S.L., and V.M.; Methodology, J.M.R., Z.V., and R.F.; Resources, J.M.R., M.G., and M.K.; Supervision, J.M.R. and M.G.; Visualization, R.F.; Writingoriginal draft, R.F., Z.V., and V.M.; Writingreview and editing, Z.V., J.M.R., M.K., V.M., M.G., R.F.

The author(s) declare that financial support was received for the research and/or publication of this article. This work was funded by the EU’s NextGenerationEU through the Recovery and Resilience Plan for Slovakia under the project No. 09I02-03-V01-00021. Further support was received from the Slovak Grant Agency KEGA (009UPJŠ-4/2023) and VEGA (1/0498/23). An Action AustriaSlovakia grant (MPC-2024–04138) administered by SAIA, n. o. and OeAD-GmbH, funded a research stay at the University of Vienna, Austria.

The authors declare no competing financial interest.

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Supplementary Materials

ao6c05106_si_001.pdf (566.4KB, pdf)

Data Availability Statement

The data presented in this study can be provided by the authors upon reasonable request.

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