Lichen Secondary Metabolites Research Articles

STABILIZATION OF POLYETHYLENE BY NATURAL FILLERS AND THEIR EXTRACTS

The possibility of using natural fillers, as well as their extracts as antioxidant additives for polyethylene, was studied. Dried and shredded buckwheat husk sowing (Fagopyrum esculentum), carposome of crab-of-the-woods (Laetiporus sulphureus), carposome of chaga mushroom (Inonotus obliquus), thallus of lichen of oakmoss (Everonia prunastri) were used as natural fillers. Polymer films were made from mixtures of powders of low-pressure polyethylene and natural fillers (the first type of samples) or from a mixture of polyethylene powder and an extract of natural fillers (the second type of samples) by thermal pressing. The degree of oxidation of polyethylene films was controlled by IR spectroscopy.
 The paper shows that the stabilization of the polyethylene structure proved to be the most effective when used as an additive to the polymer of an extract of the oakmoss (Evernia prunastri). The result is attributed to the presence of secondary lichen metabolites in the extract. The introduction of dry residues of natural fillers proved to be ineffective for polymer stabilization.

Incomplete degradation of lichen usnic acid and atranorin in Svalbard reindeer (Rangifer tarandus platyrhynchus)

Previous studies of Eurasian tundra reindeer (Rangifer tarandus tarandus) in Norway indicate that their rumen microbiota play a key role in degrading lichen secondary metabolites. We investigated the presence of usnic acid and atranorin in faecal samples from Svalbard reindeer (R. tarandus platyrhynchus). Samples were collected in Bolterdalen valley together with vegetation samples from the study site. The mesic tundra in this area was dominated by vascular plants (59% of vegetation cover). Bryophytes (16%) and lichens (25%) were also present. Qualitative and quantitative analyses of usnic acid and atranorin in lichen and faeces samples were performed using high-performance liquid chromatography. Contents of atranorin averaged 12.49 ± 0.41 mg g–1in the thalli of Stereocaulon alpinum, while the average level of usnic acid was lowest in Cladonia mitis (12.75 ± 2.86 mg g–1) and highest in Flavocetraria cucullata (34.87 ± 0.47 mg g–1). Atranorin and usnic acid were detected in the faecal samples, averaging 0.41 ± 0.53 and 0.74 ± 1.11 (mean ± SD) mg g–1 dry matter, respectively. The presence of lichen secondary compounds in faeces from Svalbard reindeer shows that lichens are indeed included in their diet, although probably in small amounts because of depleted pastures. Contrary to previous findings in reindeer on mainland Norway, atranorin and usnic acid are not completely degraded or absorbed in Svalbard reindeer. To elucidate the mechanisms behind detoxification of lichen secondary compounds in reindeer, more research is needed on their respective rumen microbiomes and digestive enzymes.

Cytotoxic, apoptotic and cell migration inhibitory effects of atranorin on SPC212 mesothelioma cells

Objective: To investigate the effects of atranorin, a lichen secondary metabolite, on SPC212 malignant mesothelioma cells in vitro. Methods: SPC212 malignant mesothelioma cell line was used. 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay was used to evaluate cytotoxic effects of atranorin and cisplatin at 24, 48 and 72 h. Hematoxylin-eosin staining and 4’,6-diamidino-2-phenylindole, dihydrochloride staining were used for determining cell and nucleus morphology, respectively. Wound healing assay was used for investigating cell migration. The xCELLigence real-time cell analysis system was used for determining cell proliferation. Results: Atranorin at 5-450 μΜ decreased cell viability at 24, 48 and 72 h. IC50 values of atranorin were 300.94, 292.6 and 278.02 μM at 24, 48 and 72 h, respectively; meanwhile, the IC50 values of cisplatin were 128.00, 34.37 and 17.05 μM at 24, 48 and 72 h, respectively. Furthermore, atranorin disrupted cell and nuclear morphology with increasing concentrations. Atranorin significantly reduced cell migration by 38%, 37% and 35% at 300, 250 and 200 μM, respectively (P Conclusions: Atranorin has cytotoxic, antiproliferative, apoptotic and cell migration inhibitory effects on SPC212 malignant mesothelioma cancer cells.

Physiological responses of Xanthoria parietina to longterm copper excess: Role of the extracellular secondary metabolite parietin

Xanthoria parietina is a common lichen that is widespread around the world. Tolerance of heavy metal pollution in this lichen is well known. Xanthoria parietina contains the secondary metabolite parietin, which protects the photobiont from high UV- radiation. Secondary metabolites of lichens have been found to form complexes with metal cations. In a long-term experiment (lasting 8 weeks), we tested the resistance of X. parietina with and without the secondary metabolite parietin against two concentrations of Cu (50 ?M and 500 ?M). Removal of parietin did not affect the measured physiological parameters. However, it caused higher accumulation of intracellular Cu. De novo synthesis of parietin in the lichen thallus after removal of the metabolite was not observed. Physiological parameters (chlorophyll a fluorescence, chlorophyll a integrity, content of soluble proteins, ergosterol levels, TBARS production) were affected by the higher dose of Cu during the long-term experiment. It seems that the secondary metabolite parietin does not protect the lichen against Cu excess.

Tumidulin, a Lichen Secondary Metabolite, Decreases the Stemness Potential of Colorectal Cancer Cells.

Lichens produce various unique chemicals that are used in the pharmaceutical industry. To screen for novel lichen secondary metabolites that inhibit the stemness potential of colorectal cancer cells, we tested acetone extracts of 11 lichen samples collected in Chile. Tumidulin, isolated from Niebla sp., reduced spheroid formation in CSC221, DLD1, and HT29 cells. In addition, mRNA expressions and protein levels of cancer stem markers aldehyde dehydrogenase-1 (ALDH1), cluster of differentiation 133 (CD133), CD44, Lgr5, and Musashi-1 were reduced after tumidulin treatment. Tumidulin decreased the transcriptional activity of the glioma-associated oncogene homolog zinc finger protein (Gli) promoter in reporter assays, and western blotting confirmed decreased Gli1, Gli2, and Smoothened (SMO) protein levels. Moreover, the tumidulin activity was not observed in the presence of Gli and SMO inhibitors. Together, these results demonstrate for the first time that tumidulin is a potent inhibitor of colorectal cancer cell stemness.

Response of secondary metabolites to Cu in the Cu-hyperaccumulator lichen Stereocaulon japonicum.

Lichen secondary metabolites are known to be associated with heavy metal uptake and tolerance in lichens. Understanding the relationship between their secondary metabolites and heavy metals in them is important for clarifying the mechanisms of their heavy metal accumulation and tolerance. To determine the relationships between the concentrations of secondary metabolites and Cu in the Cu-hyperaccumulator lichen Stereocaulon japonicum and to clarify its response to Cu, we collected Cu-contaminated and uncontaminated samples of the lichen and determined relative concentrations of secondary metabolites and concentrations of Cu, K, glucose, and sugar alcohols in them. We found significant negative correlations between the relative concentrations of secondary metabolites-atranorin and stictic acid-and the concentration of Cu. These negative correlations can be interpreted in one of two ways: (a) S. japonicum itself reduced the relative concentrations of secondary metabolites in response to the increase of Cu concentration or (b) its carbon and energy metabolism was damaged by Cu stress, resulting in the reduction of the relative concentrations of secondary metabolites. The analysis of K, glucose, and sugar alcohols showed no effect of Cu on these concentrations, which means that the carbon and energy metabolism was not damaged by Cu stress. Therefore, the negative correlations can be interpreted that S. japonicum itself reduced the relative concentrations of secondary metabolites with the increase of Cu concentration. These findings provide a deeper understanding of the response of secondary metabolites to Cu in the lichen.

Secondary metabolites from lichen as potent inhibitors of advanced glycation end products and vasodilative agents

Secondary metabolites from lichens are known for exhibiting various biological effects such as anti-inflammatory, antioxidant and antibacterial activities. Despite this wide range of reported biological effects, their impact on the formation of advanced glycation end products (AGEs) remains vastly unexplored. The latter are known contributors to lifestyle and age-related diseases such as Alzheimer and Parkinson. Moreover, the development of atherosclerosis and arterial stiffness is causally linked to the formation of AGEs. With this in mind, the present work evaluated the inhibitory effects of secondary lichen metabolites on the formation of pentosidine-like AGEs' by using an in vitro, Maillard reaction based, fluorescence assay. Overall, thirty-seven natural and five synthetically modified compounds were tested, eighteen of which exhibiting IC50 values in the range of 0.05 to 0.70 mM. This corresponds to 2 to 32 fold of the inhibitory activity of aminoguanidine. Targeting one major inhibiting mechanism of AGEs formation, all compounds were additionally evaluated on their radical scavenging capacities in an DPPH assay. Furthermore, as both AGEs' formation and hypertension are major risk factors for atherosclerosis, compounds that were available in sufficient amounts were also tested for their vasodilative effects. Overall, and though some of the active compounds were previously reported cytotoxic, present results highlight the interesting potential of secondary lichen metabolites as anti-AGEs and vasodilative agents.

Inhibitory Effect of New Semisynthetic Usnic Acid Derivatives on Human Tyrosyl-DNA Phosphodiesterase 1.

Usnic acid, a lichen secondary metabolite produced by a whole number of lichens, has attracted the interest of researchers owing to its broad range of biological activity, including antiviral, antibiotic, anticancer properties, and it possessing a certain toxicity. The synthesis of new usnic acid derivatives and the investigation of their biological activity may lead to the discovery of compounds with better pharmacological and toxicity profiles. In this context, a series of new usnic acid derivatives comprising a terpenoid moiety were synthesized, and their ability to inhibit the catalytic activity of the human DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 was investigated. The most potent compounds (15A, 15B, 15G: , and 16A, 16B, 16G: ) had IC50 values in the range of 0.33 - 2.7 µM. The inhibitory properties were mainly dependent on the flexibility and length of the terpenoid moiety, but not strongly dependent on the configuration of the asymmetric centers. The synthesized derivatives showed low cytotoxicity against human cell lines in an MTT assay. They could be used as a basis for the development of more effective anticancer therapies when combined with topoisomerase 1 inhibitors.

The role of usnic acid-induced apoptosis and autophagy in hepatocellular carcinoma.

Usnic acid (UA) is a multifunctional bioactive lichen secondary metabolite with potential anti-cancer properties. Although the promising therapeutic effects of UA have been investigated in different cancer cell lines, the mechanism driving UA-induced cell death has yet to be elucidated. As the type of cell death (apoptosis or autophagy) induced by UA may vary depending on the cancer cell type, we first studied the cytotoxic effects of UA in HEPG2 (HBV(-)) and SNU-449(HBV(+)) hepatocellular carcinoma (HCC) cell lines. HCC cell viability was considerably reduced in a dose-dependent manner at 12, 24, and 48 h after treatment with UA ( p < 0.05). However, SNU-449 cells were more sensitive to UA than HEPG2 cells. UA also induced apoptotic cell death in HCC cells with cell cycle arrest at G0/G1 and G2/M phase depending on the genetic profile of each cell type. On the other hand, we observed acidic vesicular organelles in HCC cells after 36 h of UA treatment. Taken together, these findings suggest that UA stimulates apoptosis and autophagy in HEPG2 and SNU-449 cells without damaging normal control cells. Thus, UA might be a potential therapeutic compound for HCC treatment. However, there is a need for further studies investigating the death-promoting or preventing roles for autophagy and the molecular signaling mechanisms induced by UA treatment.

Relationship between lichen species composition, secondary metabolites and soil pH, organic matter, and grain characteristics in Manitoba

Many lichen secondary metabolites have functions related to the environmental conditions of lichen habitats but few studies have compared soil characteristics with lichen species composition or their secondary products. The goal of this study was to investigate the relationship between soil characteristics with lichen species composition and secondary metabolites. Five locations were chosen in Manitoba, each with five sites (transects), and each transect with five quadrats (1 m × 1 m). All species were collected from each of the quadrats, presence of secondary metabolites was determined by thin layer chromatography, and soil characteristics were examined. The results revealed that rounded sand grains were significantly higher in southeastern Manitoba than in other locations, corresponding to a distinct species composition. Angular grains were significantly higher in northern locations, corresponding to a different group of species. Some of the significant relationships between soil characteristics and secondary metabolites include correlations of atranorin with pH, organic matter, and sand content; fumarprotocetraric acid with organic matter and sand content; and usnic acid with pH and organic matter. A better understanding of the role of lichens with respect to soil characteristics will be important for improving soil stabilization in land reclamation.

Usnic acid, as a biotic factor, changes the ploidy level in mosses.

Lichens and mosses often share the same environmental conditions where they compete for substrate and other essential factors. Lichens use secondary metabolites as allelochemicals to repel surrounding plants and potential rivals. In mosses, endoreduplication leads to the occurrence of various ploidy levels in the same individual and has been suggested as an adaptation to abiotic stresses. Here, we show that also biotic factors such as usnic acid, an allelochemical produced by lichens, directly influenced the level of ploidy in mosses. Application of usnic acid changed the nuclei proportion and significantly enhanced the endoreduplication index in two moss species, Physcomitrella patens and Pohlia drummondii. These investigations add a new aspect on secondary metabolites of lichens which count as biotic factors and affect ploidy levels in mosses.

The anti-cancer efficacies of diffractaic, lobaric, and usnic acid: In vitro inhibition of glioma.

Glioblastoma multiforme (GBM) shows the most aggressive invasion among primary brain tumors. In spite of the standard therapy methods such as surgery, radiotherapy, and chemotherapy, the mortalities are high in GBM patients owing to side effects. Some lichen secondary metabolites that have many bioactive functions exhibited anti-cancer efficacy toward many cancer types. The present study was undertaken to investigate proliferation change, oxidative status and DNA damage potentials of human U87MG-GBM, and primary rat cerebral cortex (PRCC) cells exposed to three lichen secondary metabolites. Different concentrations of lichen secondary metabolites including diffractaic acid (DA), lobaric acid (LA), and (+)-usnic acid (UA) were used for the treatments. PRCC cells were obtained from Sprague Dawley® rats. U87MG cell line was preferred as GBM cells. The results showed that lactate dehydrogenase and 8-hydroxy-2'-deoxyguanosine levels increased in PRCC and U87MG cells in a clear dose-dependent manner. Inhibitory concentration 50% (IC50) values of LA, DA, and UA were calculated as 9.08, 122.26, 132.69 mg/L for PRCC cells and 5.77, 35.67, 41.55 mg/L for U87MG cells, respectively. Concentration of 10 mg/L of DA and UA demonstrated high anti-oxidant capacity on healthy PRCC cells. Overall, obtained data indicated that LA was highly toxic on GBM and PRCC cells. However, DA and then UA had high anti-oxidant capacity on PRCC cells. These results suggest that further studies that will be held on LA may play a critical role in GBM treatment.

Usnic Acid: Potential Role in Management of Wound Infections.

Usnic acid (UA) is a secondary lichen metabolite extensively studied for the broad variety of biological features. The most interesting property of UA is its antimicrobial activity against Gram-positive bacteria growing either in planktonic or in biofilm mode. In this chapter, the most relevant studies assessing usnic acid activity against microbial biofilms have been summarized and the potential role of UA in the management of biofilm-based wound infections has been critically discussed. Additionally, an overview of the main strategies adopted so far to reduce drug toxicity and increase bioavailability is given in the perspective of a safe use of UA in the clinical management of infected wounds.

Atranorin - An Interesting Lichen Secondary Metabolite.

Atranorin, a compound with the depside structure, is one of the most common lichen secondary metabolites, characteristic for numerous lichen families but rarely found in some mosses and higher plants. Over the years various biological properties of atranorin were examined. This review summarizes the studies on atranorin, focusing on a number of biological activities in different fields. The literature describes anti-inflammatory, analgesic, as well as wound healing, antibacterial, antifungal, cytotoxic, antioxidant, antiviral, and immunomodulatory activities of the depside. Furthermore, lack of toxicity of atranorin was confirmed in the animals' in vivo assays. In conclusion, atranorin seems to be an interesting lichen substance, which needs to be investigated in more detail in order to allow further applications, e.g. in pharmacy, medicine or cosmetology.

Application of high-performance liquid chromatography to the determination of the concentration of lichen secondary metabolites

Preparative isolation of aromatic metabolites from lichens of the Cladonia genus (C. stellaris, C. arbuscula, C. amaurocraea, and C. rangiferina) growing in Central Yakutia was carried out. Identification by IR, UV, and time-of-flight mass spectrometry has shown that the isolated compounds belong to the group of lichen substances. It was shown that the component composition of the studied lichens corresponds to the previously described one. At the same time, the concentration of perlatolic and barbatic acids in C. stellaris and C. amaurocrae lichens, respectively, growing in Central Yakutia found by HPLC is higher than in similar types of temperate climatic zones.

STUDY OF THE COMPOSITION AND PROPERTIES OF SECONDARY METABOLITES OF THE LICHEN USNEA BARBATA

In the modern society value of many biological resources remains underestimated. Lichens are one of the unique poorly studied bioresources. These are amazing organisms, formed by symbiosis of algae and fungus. Due to this combination, lichens have a number of unique properties.In this article the results of study the composition of biologically active secondary lichen metabolites of the Usnea barbata lichen. Antioxidant activity and antimicrobial properties of dry lichen extract against bacteria Bac. Subtilis. The conditions for extraction of biologically active metabolites using solvent systems such as water, water-ethanol mixtures with ethanol content of 40 and 70%, 1,4-dioxane and a mixture of 1,4-dioxane and water (1: 1) were selected. With use of the modern analytical methods, the composition of secondary metabolites in the extracts was studied. By the method of spectroscopy in the UV and visible region of the spectrum to determine the content usnic acid, which is 16,2 (solvent - water) to 60,0 (1,4-dioxane) mg/100 ml of extract.It is shown that the dioxane extract has pronounced atioxidant properties. The active substances (in terms of usnic acid) of the dry extract of lichen Usnea barbata are able to inhibit the growth of bacteria Bac. Subtilis.

Abstract 4209: Inhibitory activity of lichen secondary metabolite, physciosporin, against lung cancer cell motility

Abstract Lichens produce various unique chemicals that can be used for pharmaceutical purposes. To screen for novel lichen secondary metabolites showing inhibitory activity against lung cancer cell motility, we tested acetone extracts of 13 lichen samples collected in Chile. Physciosporin, isolated from Pseudocyphellaria coriacea (Hook f. &amp; Taylor) D.J. Galloway &amp; P. James, was identified as an effective compound and showed significant inhibitory activity in migration and invasion assays against human lung cancer cells. Physciosporin treatment reduced both protein and mRNA levels of N-cadherin with concomitant decreases in the levels of epithelial-mesenchymal transition markers such as snail and twist. Physciosporin also suppressed KITENIN (KAI1 C-terminal interacting tetraspanin)-mediated AP-1 activity in both the absence and presence of epidermal growth factor stimulation. Quantitative real-time PCR analysis showed that the expression of the metastasis suppressor gene, KAI1, was increased while that of the metastasis enhancer gene, KITENIN, was dramatically decreased by physciosporin. Particularly, the activity of 3'-untranslated region of KITENIN was decreased by physciosporin. Moreover, Cdc42 and Rac1 activities were decreased by physciosporin. These results demonstrated that the lichen secondary metabolite, physciosporin, inhibits lung cancer cell motility through novel mechanisms of action. Citation Format: Yi Yang, Young Hyun Yu, Hyung-Ho Ha, Kyung-Hwa Lee, Kyung-Sub Moon, Kyung Keun Kim, Hangun Kim. Inhibitory activity of lichen secondary metabolite, physciosporin, against lung cancer cell motility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4209. doi:10.1158/1538-7445.AM2017-4209

Morphological, Secondary Metabolite and ITS (rDNA) Variability within Usnic Acid-Containing Lichen Thalli of Xanthoparmelia Explored at the Local Scale of Rock Outcrop in W-Alps.

Lichen secondary metabolites (LSMs) are regarded with interest for valuable biological properties, but chemical variability among/within lichen taxa has been only fragmentarily characterized by advanced analytical techniques. Knowledge of variability at a local geographic scale has been particularly neglected, while it should address the collection of chemically homogeneous materials to test and exploit LSMs. Here we evaluated the chemical variability of 48 Xanthoparmelia specimens from two rock outcrops in Western Italian Alps, representative of nine morphotypes and sixteen rDNA ITS haplotypes. Qualitative and quantitative analyses were performed by HPLC-DAD-ESI-MS2 and UPLC-HDR-DAD, respectively, and revealed the occurrence of 18 LSMs. Chemical partition allowed distinguishing six chemical groups, only partially overlapping with distinct morphotypes and three divergent haplotype groups, which, overall, accounted for the co-occurrence of different taxa only in part identifiable with species described for Europe. Some morphotypes were variable in presence and concentration of LSMs, and chemical divergences also characterized single ITS haplotypes. Accordingly, the collection of chemically homogeneous materials, even at a local scale, may be not properly addressed by morphological features and ITS barcoding, and should be confirmed by a specimen-level chemical characterization.

SOS response in bacteria: Inhibitory activity of lichen secondary metabolites against Escherichia coli RecA protein

BackgroundRecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown. PurposeTwenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens. MethodsThe ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition vs. ATP and vs. ssDNA, was evaluated. ResultsOnly nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC50) calculated for these compounds were ranging from 14.2µM for protolichesterinic acid to 42.6µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were confirmed by molecular modelling binding predictions which shows that epiphorellic acid is expected to bind the ssDNA site into the L2 loop of RecA protein. ConclusionIn this paper the first RecA ssDNA binding site ligand is described. Our study sets epiphorellic acid as a promising hit for the development of more effective RecA inhibitors. In our drug discovery approach, natural products in general and lichen in particular, represent a successful source of active ligands and structural diversity.

Reducing Virulence and Biofilm of Pseudomonas aeruginosa by Potential Quorum Sensing Inhibitor Carotenoid: Zeaxanthin.

Pseudomonas aeruginosa can regulate its virulence gene expressions by using a signal system called quorum sensing. It is known that inhibition of quorum sensing can block biofilm formation and leave the bacteria defenseless. Therefore, it is necessary to determine natural sources to obtain potential quorum sensing inhibitors. This study aims to investigate an alternative treatment approach by utilizing the carotenoid zeaxanthin to reduce the expressions of P. aeruginosa virulence factors through quorum sensing inhibition. The inhibition potential of zeaxanthin was determined by in silico screening from a library of 638 lichen metabolites. Fluorescent monitor strains were utilized for quorum sensing inhibitor screens, and quantitative reverse-transcriptase PCR assay was performed for evaluating gene expression. Results indicate that zeaxanthin is a better inhibitor than the lichen secondary metabolite evernic acid, which was previously shown to be capable of inhibiting P. aeruginosa quorum sensing systems.