Mycobiont-specific primers facilitate the amplification of mitochondrial small subunit ribosomal DNA: a focus on the lichenized fungal genus Melanelia (Ascomycota, Parmeliaceae) in Iceland.

The fungal mitochondrial small subunit (mtSSU) ribosomal DNA is one of the most commonly used loci for phylogenetic analysis of lichen-forming fungi, but their primer specificity to mycobionts has not been evaluated. The current study aimed to design mycobiont-specific mtSSU primers and highlights their utility with an example from the saxicolous lichen-forming fungal genus Melanelia Essl. in Iceland. The study found a 12.5% success rate (3 out of 24 specimens with good-quality mycobiont mtSSU sequences) using universal primers (i.e. mrSSU1 and mrSSU3R), not including off-target amplification of environmental fungi, e.g. Cladophialophoracarrionii and Lichenotheliaconvexa. New mycobiont-specific primers (mt-SSU-581-5' and mt-SSU-1345-3') were designed by targeting mycobiont-specific nucleotide sites in comparison with environmental fungal sequences, and assessed for mycobiont primer specificity using in silico PCR. The new mycobiont-specific mtSSU primers had a success rate of 91.7% (22 out of 24 specimens with good-quality mycobiont mtSSU sequences) on the studied Melanelia specimens. Additional testing confirmed the specificity and yielded amplicons from 79 specimens of other Parmeliaceae mycobiont lineages. This study highlights the effectiveness of designing mycobiont-specific primers for studies on lichen identification, barcoding and phylogenetics.

Novel methods to characterise spatial distribution and enantiomeric composition of usnic acids in four Icelandic lichens.

Usnic acid is an antibiotic metabolite produced by a wide variety of lichenized fungal lineages. The enantiomers of usnic acid have been shown to display contrasting bioactivities, and hence it is important to determine their spatial distribution, amounts and enantiomeric ratios in lichens to understand their roles in nature and grasp their pharmaceutical potential. The overall aim of the study was to characterise the spatial distribution of the predominant usnic acid enantiomer in lichens by combining spatial imaging and chiral chromatography. Specifically, separation and quantification of usnic acid enantiomers in four common lichens in Iceland was performed using a validated chiral chromatographic method. Molecular dynamics simulation was carried out to rationalize the chiral separation mechanism. Spatial distribution of usnic acid in the lichen thallus cross-sections were analysed using Desorption Electrospray Ionization-Imaging Mass Spectrometry (DESI-IMS) and fluorescence microscopy. DESI-IMS confirmed usnic acid as a cortical compound, and revealed that usnic acid can be more concentrated around the algal vicinity. Fluorescence microscopy complemented DESI-IMS by providing more detailed distribution information. By combining results from spatial imaging and chiral separation, we were able to visualize the distribution of the predominant usnic acid enantiomer in lichen cross-sections: (+)-usnic acid in Cladonia arbuscula and Ramalina siliquosa, and (-)-usnic acid in Alectoria ochroleuca and Flavocetraria nivalis. This study provides an analytical foundation for future environmental and functional studies of usnic acid enantiomers in lichens.

Ethnopharmacology of the club moss subfamily Huperzioideae (Lycopodiaceae, Lycopodiophyta): A phylogenetic and chemosystematic perspective.

ETHNOPHARMACOLOGICAL RELEVANCE: The most speciose subfamily Huperzioideae (Lycopodiaceae, Lycopodiophyta) contains about 276 species, and some (ca. 20 species) have traditionally been used for the treatment of e.g., dementia, rheumatism and traumatic injury. Ethnopharmacological studies have also contributed to the development of huperzine A as a drug lead, a compound first isolated from the club moss Huperzia serrata (Thunb. ex Murray) Trevis. AIM OF THE REVIEW: This review, with a phylogenetic and chemosystematic perspective, intends to highlight plant identification challenges in these taxa with examples from club moss phytochemical and ethnopharmacological studies, as these lead to data inconsistency and confusion. We suggest that future studies should include more details on plant identification including for example plant specimen images and DNA barcoding data. An integrative approach combining DNA barcoding and chemical fingerprinting is also introduced. MATERIALS AND METHODS: Literature concerning ethnopharmacology and chemosystematics of Huperzioideae club mosses was searched from databases, e.g. PubMed, Web of Science, SciFinder, etc. Plant names were retrieved from original publications, and compared with up-to-date taxonomic and phylogenetic status. Ethnobotanical uses and herbal preparations were summarized. Production of certain pharmaceutically interesting compounds, such as the alkaloid huperzine A, was explored in a phylogenetic context. RESULTS: Most traditionally used club mosses are associated with psychoactivity, followed by medicinal uses against rheumatism and traumatic injury. Herbs are often prepared as infusions, decoctions or tinctures, and this implies importance of water- or aqueous-alcohol-soluble substances, such as alkaloids. Most ethnopharmacological papers on club mosses need to update or correct plant names according to recent taxonomic nomenclature, and there are still a number of unidentified species with traditional use. Advanced LC-MS chemical profiling techniques, enable distinction of genotypes of the same species as well as annotation of potential chemotaxonomic markers. In combination with DNA barcoding, chemosystematics could also help us select plant taxa with higher pharmaceutical potential. Caution should be taken when interpreting bioassay results, in terms of compounds or extract preparation and bioassay standardization. CONCLUSION: Huperzioideae club mosses have interesting pharmaceutical potential supported by ethnopharmacological investigations. Bioprospecting of these plants should be preceded by careful plant identification to produce consistent and reproducible data. We expect that DNA barcoding and LC-MS-based chemical fingerprinting could facilitate and improve ethnopharmaceutical studies in selection of club moss taxa.

Exopolysaccharides from Cyanobacterium aponinum induce a regulatory dendritic cell phenotype and inhibit SYK and CLEC7A expression in dendritic cells, T cells and keratinocytes.

Regular bathing in the Blue Lagoon has beneficial effects on psoriasis. Previously, we showed that exopolysaccharides (EPS-Ca) secreted by Cyanobacterium aponinum, a dominating organism in the Blue Lagoon, increased IL-10 secretion by human dendritic cells (DCs). In addition, co-culturing allogeneic CD4+ T cells with DCs matured in the presence of EPS-Ca increased differentiation of T cells into T regulatory cells at the cost of the disease inducing Th17 cells. In the present study, EPS-Ca increased the proportion of DCs expressing CD141, a surface molecule linked to regulatory DCs, and the CD141+ cells secreted more IL-10 than the CD141- cells. EPS-Ca decreased T cell secretion of IL-17, IL-13 and IL-10 and the proportion of T cells expressing the activation marker CD69 that has also been linked to lymphocyte retention. In addition, EPS-Ca reduced keratinocyte secretion of CCL20 and CXCL10, chemokines implicated in recruitment of inflammatory cells. EPS-Ca decreased DC expression of Dectin-1/CLEC7A and SYK, keratinocyte expression of CLEC7A, SYK and CAMP (the gene for LL37), and T cell expression of phosphorylated Zap70. These results indicate that EPS-Ca may induce a regulatory phenotype of DCs, T cells that are less active/inflammatory and less prone to being retained in the skin, and keratinocytes that induce less recruitment of inflammatory cells to the skin and that these effects may be mediated by the effects of EPS-Ca on CLEC7A and SYK. Overall the results indicate that EPS-Ca may be involved in the beneficial effects psoriasis patients experience when bathing in the Blue Lagoon.

Infraspecific Variation of Huperzine A and B in Icelandic Huperzia selago Complex.

The alkaloids huperzine A and huperzine B were originally isolated from the Chinese club moss Huperzia serrata. They are known inhibitors of acetylcholinesterase, and especially huperzine A shows pharmaceutical potential for the treatment of Alzheimer's disease. Its supply heavily relies on natural plant sources belonging to the genus Huperzia, which shows considerable interspecific huperzine A variations. Furthermore, taxonomic controversy remains in this genus, particularly in the Huperzia selago group. With focus on Icelandic H. selago taxa, we aimed to explore the relatedness of Huperzia species using multi-locus phylogenetic analysis, and to investigate correlations between huperzine A contents, morphotypes, and genotypes. Phylogenetic analysis was performed with five chloroplastic loci (the intergenic spacer between the photosystem II protein D1 gene and the tRNA-His gene, maturase K, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, tRNA-Leu, and the intergenic spacer region between tRNA-Leu and tRNA-Phe). Huperzine A and huperzine B contents were determined using an HPLC-UV method. The phylogenetic analysis suggests that previously proposed Huperzia appressa and Huperzia arctica should not be considered species, but rather subspecies of H. selago. Three genotypes of Icelandic H. selago were identified and presented in a haplotype networking diagram. A significantly (p < 0.05) higher amount of huperzine A was found in H. selago genotype 3 (264 - 679 µg/g) than genotype 1 (20 - 180 µg/g), where the former shows a typical green and reflexed "selago" morphotype. The huperzine A content in genotype 3 is comparable to Chinese H. serrata and a good alternative huperzine A source. Genotype 2 contains multiple morphotypes with a broad huperzine A content (113 - 599 µg/g). The content of huperzine B in Icelandic taxa (6 - 13 µg/g) is much lower than that in Chinese H. serrata (79 - 207 µg/g).

Impact of dibenzocyclooctadiene lignans from Schisandra chinensis on the redox status and activation of human innate immune system cells.

Redox signaling has been established as an essential component of inflammatory responses, and redox active compounds are of interest as potential immunomodulatory agents. Dibenzocyclooctadiene lignans isolated from Schisandra chinensis, a medicinal plant with widespread use in oriental medicine, have been implicated to possess immunomodulatory properties but their effects on the human innate immune system cells have not been described. In this contribution, data are presented on the impact of schisandrin, schisandrin B and schisandrin C on human monocytic cell redox status, as well as their impact on dendritic cell maturation and T cell activation capacity and cytokine production. In THP-1 cells, levels of intracellular reactive oxygen species (ROS) were elevated after 1 h exposure to schisandrin. Schisandrin B and schisandrin C decreased cellular glutathione pools, which is a phenotype previously reported to promote anti-inflammatory functions. Treatment of human primary monocytes with the lignans during their maturation to dendritic cells did not have any effect on the appearance of surface markers HLA-DR and CD86 but schisandrin B and schisandrin C suppressed the secretion of cytokines interleukin (IL)-6, IL-10 and IL-12 by the mature dendritic cells. Dendritic cells maturated in presence of schisandrin C were further cocultured with naïve CD4+ T cells, resulting in reduced IL-12 production. In THP-1 cells, schisandrin B and schisandrin C reduced the IL-6 and IL-12 production triggered by E. coli lipopolysaccharide and IL-12 production induced by an infection with Chlamydia pneumoniae. In conclusion, the studied lignans act as immunomodulatory agents by altering the cytokine secretion, but do not interfere with dendritic cell maturation. And the observed effects may be associated with the ability of the lignans to alter cellular redox status.

Authentication of Iceland Moss (Cetraria islandica) by UPLC-QToF-MS chemical profiling and DNA barcoding.

The lichen Cetraria islandica or Iceland Moss is commonly consumed as tea, food ingredients (e.g. in soup or bread) and herbal medicines. C. islandica, which has two chemotypes, can be difficult to distinguish from the sister species Cetraria ericetorum. They are collectively referred to as the Cetraria islandica species complex. This study aimed to use an UPLC-QToF-MS chemical profiling together with DNA barcoding to distinguish species and chemotypes of the C. islandica species complex. Our results show that the two chemotypes of C. islandica are clearly distinguishable from each other and from C. ericetorum by the chemometric approach. The RPB2 barcode was able to differentiate C. islandica from C. ericetorum with a barcode gap, but the widely used nrITS barcode failed. Neither of them could discriminate chemotypes of C. islandica. In conclusion, this integrative approach involving chemical profiling and DNA barcoding could be applied for authentication of Iceland Moss materials.

Secondary metabolites from cetrarioid lichens: Chemotaxonomy, biological activities and pharmaceutical potential.

BACKGROUND: Lichens, as a symbiotic association of photobionts and mycobionts, display an unmatched environmental adaptability and a great chemical diversity. As an important morphological group, cetrarioid lichens are one of the most studied lichen taxa for their phylogeny, secondary chemistry, bioactivities and uses in folk medicines, especially the lichen Cetraria islandica. However, insufficient structure elucidation and discrepancy in bioactivity results could be found in a few studies. PURPOSE: This review aimed to present a more detailed and updated overview of the knowledge of secondary metabolites from cetrarioid lichens in a critical manner, highlighting their potentials for pharmaceuticals as well as other applications. Here we also highlight the uses of molecular phylogenetics, metabolomics and ChemGPS-NP model for future bioprospecting, taxonomy and drug screening to accelerate applications of those lichen substances. CHAPTERS: The paper starts with a short introduction in to the studies of lichen secondary metabolites, the biological classification of cetrarioid lichens and the aim. In light of ethnic uses of cetrarioid lichens for therapeutic purposes, molecular phylogeny is proposed as a tool for future bioprospecting of cetrarioid lichens, followed by a brief discussion of the taxonomic value of lichen substances. Then a delicate description of the bioactivities, patents, updated chemical structures and lichen sources is presented, where lichen substances are grouped by their chemical structures and discussed about their bioactivity in comparison with reference compounds. To accelerate the discovery of bioactivities and potential drug targets of lichen substances, the application of the ChemGPS NP model is highlighted. Finally the safety concerns of lichen substances (i.e. toxicity and immunogenicity) and future-prospects in the field are exhibited. CONCLUSION: While the ethnic uses of cetrarioid lichens and the pharmaceutical potential of their secondary metabolites have been recognized, the knowledge of a large number of lichen substances with interesting structures is still limited to various in vitro assays with insufficient biological annotations, and this area still deserves more research in bioactivity, drug targets and screening. Attention should be paid on the accurate interpretation of their bioactivity for further applications avoiding over-interpretations from various in vitro bioassays.

The Genus Diphasiastrum and Its Lycopodium Alkaloids.

The genus Diphasiastrum includes at least 23 species distributed primarily across the northern temperate and subarctic areas of the world. These plants produce an array of lycopodium alkaloids, and some species such as Diphasiastrum complanatum have been used in traditional medicine for ages for various conditions. Hybridization is common in this group of plants and they have always been a challenge for taxonomists and other scientists studying them. To date, 11 Diphasiastrum species have been reported to produce lycopodium alkaloids. In this review, reported alkaloids and their distribution patterns across these species along with taxonomical and bioactivity considerations are reviewed and discussed.

Dendritic cells matured in the presence of the lycopodium alkaloid annotine direct T cell responses toward a Th2/Treg phenotype.

Annotine is a lycopodane-type alkaloid isolated from the Icelandic club moss Lycopodium annotinum ssp. alpestre. Annotine does not inhibit acetylcholinesterase, as some other lycopodium alkaloids do, and other bioactivities have not been reported. The aim of this study was to determine the effects of annotine on maturation of dendritic cells (DCs) and their ability to activate allogeneic CD4(+) T cells. Human monocyte-derived DCs were matured in the absence or presence of annotine at a concentration of 1, 10 or 100 µg/ml. The effect of the annotine on maturation of the DCs was determined by measuring concentration of cytokines in culture supernatant by ELISA and expression of surface molecules by flow cytometry. DCs matured in the absence or presence of annotine at 100 µg/ml were also co-cultured with allogeneic CD4(+) T cells and concentration of cytokines in supernatants determined by ELISA and expression of surface molecules by flow cytometry. When cultured alone, DCs matured in the presence of annotine secreted less of the pro-inflammatory cytokines IL-6 and IL-23 and had a tendency toward less secretion of IL-12p40 than DCs matured in the absence of annotine. However, when DCs were matured in the presence of annotine and then co-cultured with allogeneic CD4(+) T cells they secreted more IL-12p40 and had a tendency toward secreting more IL-6 than DCs matured in the absence of annotine and then co-cultured with T cells. Allogeneic CD4(+) T cells co-cultured with DCs matured in the presence of annotine secreted more IL-13 than T cells co-cultured with DCs matured in the absence of annotine, but stimulating the DCs in the presence of annotine did not affect T cell secretion of IFN-γ and IL-17. There was also more IL-10 in co-cultures of T cells and DCs matured in the presence of annotine than in co-cultures of T cells and DCs matured in the absence of annotine. These results show that annotine increases the ability of DCs to direct the differentiation of allogeneic CD4(+) T cells toward a Th2/Treg phenotype, which may be of interest in the development of new treatments for Th1- and/or Th17-mediated inflammatory diseases.

A heteroglycan from the cyanobacterium Nostoc commune modulates LPS-induced inflammatory cytokine secretion by THP-1 monocytes through phosphorylation of ERK1/2 and Akt.

Cyanobacteria (blue-green algae) have been consumed as food and used in folk medicine since ancient times to alleviate a variety of diseases. Cyanobacteria of the genus Nostoc have been shown to produce complex exopolysaccharides with antioxidant and antiviral activity. Furthermore, Nostoc sp. are common in cyanolichen symbiosis and lichen polysaccharides are known to have immunomodulating effects. Nc-5-s is a heteroglycan isolated from free-living colonies of Nostoc commune and its structure has been characterized in detail. The aim of this study was to determine the effects of Nc-5-s on the inflammatory response of lipopolysaccharide (LPS)-stimulated human THP-1 monocytes and how the effects are mediated. THP-1 monocytes primed with interferon-γ and stimulated with LPS in the presence of Nc-5-s secreted less of the pro-inflammatory cytokine interleukin (IL)-6 and more of the anti-inflammatory cytokine IL-10 than THP-1 monocytes stimulated without Nc-5-s. In contrast, Nc-5-s increased LPS-induced secretion of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-8. Nc-5-s decreased LPS-induced phosphorylation of the extracellular regulated kinase (ERK)1/2 and Akt kinase, but did not affect phosphorylation of the p38 kinase, activation of the nuclear factor kappa B pathway, nor DNA binding of c-fos. These results show that Nc-5-s has anti-inflammatory effects on IL-6 and IL-10 secretion by THP-1 monocytes, but its effects are pro-inflammatory when it comes to TNF-α and IL-8. Furthermore, they show that the effects of Nc-5-s may be mediated through the ERK1/2 pathway and/or the Akt/phosphoinositide 3-kinase pathway and their downstream effectors. The ability of Nc-5-s to decrease IL-6 secretion, increase IL-10 secretion and moderate ERK1/2 activation indicates a potential for its development as an anti-inflammatory agent.

Marchantin A, a macrocyclic bisbibenzyl ether, isolated from the liverwort Marchantia polymorpha, inhibits protozoal growth in vitro.

In vitro anti-plasmodial activity-guided fractionation of a diethyl ether extract of the liverwort species Marchantia polymorpha, collected in Iceland, led to isolation of the bisbibenzyl ether, marchantin A. The structure of marchantin A (1) was confirmed by NMR and HREIMS. Marchantin A inhibited proliferation of the Plasmodium falciparum strains, NF54 (IC(50)=3.41µM) and K1 (IC(50)=2.02µM) and showed activity against other protozoan species Trypanosoma brucei rhodesiense, T. cruzi and Leishmania donovani with IC(50) values 2.09, 14.90 and 1.59µM, respectively. Marchantin A was tested against three recombinant enzymes (PfFabI, PfFabG and PfFabZ) of the PfFAS-II pathway of P. falciparum for malaria prophylactic potential and showed moderate inhibitory activity against PfFabZ (IC(50)=18.18µM). In addition the cytotoxic effect of marchantin A was evaluated. This is the first report describing the inhibitory effects of the liverwort metabolite marchantin A against these parasites in vitro.

Acetylcholinesterase inhibitory activity of lycopodane-type alkaloids from the Icelandic Lycopodium annotinum ssp. alpestre.

The aim of this study was to investigate structures and acetylcholinesterase inhibitory activities of lycopodane-type alkaloids isolated from an Icelandic collection of Lycopodium annotinum ssp. alpestre. Ten alkaloids were isolated, including annotinine, annotine, lycodoline, lycoposerramine M, anhydrolycodoline, gnidioidine, lycofoline, lannotinidine D, and acrifoline, as well as a previously unknown N-oxide of annotine. 1H and 13C NMR data of several of the alkaloids were provided for the first time. Solvent-dependent equilibrium constants between ketone and hemiketal form of acrifoline were determined. Conformation of acrifoline was characterized using NOESY spectroscopy and molecular modelling. The isolated alkaloids were evaluated for their in vitro inhibitory activity against acetylcholinesterase and butyrylcholinesterase. Ligand docking studies based on mutated 3D structure of Torpedo californica acetylcholinesterase provided rationale for low inhibitory activity of the isolated alkaloids as compared to huperzine A or B, which are potent acetylcholinesterase inhibitors belonging to the lycodine class. Based on the modelling studies the lycopodane-type alkaloids seem to fit well into the active site gorge of the enzyme but the position of their functional groups is not compatible with establishing strong hydrogen bonding interactions with the amino acid residues that line the binding site. The docking studies indicate possibilities of additional functionalization of the lycopodane skeleton to render potentially more active analogues.

Immunomodulating polysaccharides from the lichen Thamnolia vermicularis var. subuliformis.

Three heteroglycans Ths-4, Ths-5 and thamnolan and a beta-glucan, Ths-2, isolated from the lichen Thamnolia vermicularis var. subuliformis were tested for in vitro immunomodulating activities and shown to have various influences on the immune system. All the polysaccharides except Ths-4 caused a stimulation of rat spleen cell proliferation. In contrast, Ths-4 caused cell death early in the culture, probably due to over-stimulation. Moreover, the galactofuranomannans, Ths-4, Ths-5 and the beta-glucan Ths-2, induced rat spleen cells to secrete IL-10 significantly above background levels. In addition, Ths-4 and Ths-5 stimulated significant TNF-alpha secretion by rat peritoneal macrophages. The galactofuranomannans Ths-4 and Ths-5 have similar structures apart from the molecular weight. Thus, it may be concluded that the molecular size might influence the potency but not the pattern of activity for Ths-4 and Ths-5. The galactofuranorhamnan thamnolan had less mitogenic effect than Ths-5 and Ths-2 and neither induced IL-10 secretion by rat spleen cells nor TNF-alpha secretion by peritoneal macrophages to significant levels. This shows that thamnolan with its unusual galactofuranorhamnan structure differs from the other Thamnolia polysaccharides in its immunomodulatory activity.

Immunomodulating effects of lichen-derived polysaccharides on monocyte-derived dendritic cells.

Many naturally occurring polysaccharides from fungi and lichens have been found to have immunomodulating activity. However, the majority of these studies have focused on their effects on the innate arm of the immune system. Although dendritic cells (DCs) belong to the innate immune system, they play an important role as a bridge between the innate and the adaptive immune response. In this study, the effects of 11 chromatographically purified and well-characterised lichen polysaccharides (of different structural types) on the maturation of DCs were tested by analysing the secretion of IL-12p40 and IL-10 by human monocyte-derived dendritic cells in vitro. Four of the polysaccharides upregulated IL-10 secretion by the dendritic cells, as compared with unstimulated cells, the beta-glucans lichenan and Ths-2 and the heteroglycans Pc-4 and thamnolan. IL-12p40 secretion was significantly upregulated by the beta-glucan lichenan and the heteroglycans Pc-2, Pc-4, thamnolan and Ths-4, while the mature dendritic cells stimulated with the heteroglycan Pc-1 secreted significantly less IL-12p40 than the unstimulated cells. Proportional index (PI) was used to determine the relationship between the IL-12p40 and IL-10 secretion. The PI of all the beta-glucans, i.e. lichenan, pustulan and Ths-2, and the heteroglycan thamnolan was significantly lower than the PI observed for the unstimulated cells, which was mainly due to increased IL-10 secretion. Therefore, these polysaccharides could be considered suitable candidates in tolerance and anti-inflammatory studies, as IL-10 is one of the major cytokines involved in tolerance and anti-inflammatory responses.