The Influence of Seasonality on Secondary Metabolite Profiles and Neuroprotective Activities of Moss Hypnum cupressiforme Extracts: In Vitro and In Silico Study

Numerous representatives of mosses, including Hypnum cupressiforme, have been used to alleviate different inflammation-related conditions. However, the mode of action underlying this anti-inflammatory potential has been poorly understood. Moreover, the influence of seasonality on the chemical composition and biological activity of mosses is generally overlooked. This study aimed to investigate the influence of seasonal changes (spring, summer, and autumn) on secondary metabolite composition and biological activities of ethyl acetate H. cupressiforme extracts. Antioxidant activity was measured using β-carotene bleaching assay, while MTT, NBT, ELISA, and Griess assays were carried out to explore the anti-neuroinflammatory and neuroprotective potential of extracts. Inhibitory activities on acetylcholinesterase and tyrosinase were assessed experimentally and by docking analysis. The highest content of secondary metabolites and antioxidant activity were observed in moss during the summer. Extracts inhibited the secretion of ROS, NO, TNF-α, and IL-6, alleviating the inflammatory potential of H2O2 and LPS in microglial and neuronal cells. Strong inhibitory effects on acetylcholinesterase and tyrosinase were observed in vitro. Docking analyses revealed high-affinity interactions of secondary metabolites present in H. cupressiforme with important enzyme residues. Altogether, these results reveal the neuroprotective potential and the significance of seasonal fluctuations on secondary metabolite content and biological activities in moss H. cupressiforme.

Could Bryophagous Beetles (Coleoptera: Byrrhidae) Help Us Understand Bryophyte Taxonomy? Preferences within the Hypnum cupressiforme Hedw. Species Complex

Intrataxonomic differences in terms of angiosperm suitability for herbivorous insects stem from variables such as plant structure, palatability, and chemistry. It has not yet been elucidated whether these differences also occur in terms of the bryophyte’s suitability to bryophages. Hypnum cupressiforme Hedw. is a morphologically variable moss species frequently inhabited or fed by insects. In this investigation, we offered five morphotypes of H. cupressiforme to two bryophagous species of Byrrhidae (Coleoptera) to reveal whether the intrataxonomic variability affects beetles’ preferences. The morphotypes were offered with preserved and removed spatial structures. There were no significant differences in morphotype preferences when spatial structures were preserved, although during the daytime, the beetles moved from the flat morphotype to the usual and turgid morphotypes. The beetles preferred the turgid morphotype when the spatial structures were removed. The results suggest that the spatial structure variations in the H. cupressiforme complex are accompanied by different chemical, physiological, or microscopic morphological profiles that are recognized by the bryophagous insects. Phylogenetic and epigenetic analyses can reveal multiple differences within the H. cupressiforme complex. Their interconnection with information about the preferences of bryophagous insects can help us to elucidate which of these differences are ecologically relevant.

BRYOPHYTA AS ТЕST-ОBJECTS OF BRYOGEOCHEMICAL INDICATION OF ATMOSPHERIC FALLOUTS OF HEAVY METALS AND RADIONUCLIDES IN THE ENVIRONMENT OF EUROPE. АNALYTICAL REVIEW

The review presents the analysis of publications dedicated to problems of using of Bryobionta representatives for bryogeochemical indication and biomonitoring of heavy metals and radionuclides in the environment. Taxonomic structure of Bryobionta is briefly observed, three divisions of Bryobionta are elucidated – Anthocerotophyta, Marchantiophyta and Bryophyta. It is concluded that the most suitable moss species for biomonitoring of heavy metals and radionuclides are representatives from division Bryophyta, such as Hylocomium splendens, Pleurozium schreberi, Hypnum cupressiforme, Scleropodium purum. The mosses have been successfully used in biomonitoring of atmospheric fallout of heavy metals in the environment in Europe for 25 years. A special system of monitoring on their basis is applied in 28 countries of the continent. For the most important moss species used as test-objects of biomonitoring, significant width of their geographic distribution is shown as well as distribution on different substrates of growth (epigeious, epiphytic, epilytic). The main biological peculiarities of mosses which allow to use them for purposes of biomonitoring of heavy metals and radionuclides have been analyzed, i.e. absence of roots, that permits them to derive the main part of nutrients (and pollutants) directly from aerial fallouts – dry (dust) and wet (rain, snow), and high cation exchange capacity of their cell membranes. The most important anatomical and morphological features of three moss groups (endohydritic, ectohydritic, mixohydritic) are briefly reported, and a conclusion about the best suitability of ectohydritic moss species for bryogeochemical indication and biomonitoring of pollutants is made. Results of numerous biomonitoring studies conducted with using of widely distributed moss species in Europe in nature and anthropogenic biogeocenoses are demonstrated. Criteria to mosses as test-objects of bryogeochemical indication and biomonitoring are briefly reported. Physiological adaptations of mosses to stress emerging due to intake of significant concentrations of heavy metals to their phytomass are generalized. Requirements to sampling of moss cover for purposes of bryogeochemical indication and biomonitoring of pollutants are reported. Perspective moss species as test-objects of environmental pollution by heavy metals and radionuclides are proposed for different natural zones of Ukraine: for Polissya zone – Hylocomium splendens and Pleurozium schreberi, for Forest-Steppe zone – Hypnum cupressiforme, for Steppe zone – Tortula muralis and Bryum argenteum.

ELEMENTAL CONCENTRATIONS AND SOIL-TO-MOSS TRANSFER FACTORS OF RADIONUCLIDES IN THE ENVIRONMENT OF NORTH KOSOVO AND METOHIJA

This paper deals with investigations of elemental concentrations and soil-to-moss transfer factors of radionuclides in area of municipalities Kosovska Mitrovica and Zubin Potok. Twelve samples of soil and moss Hypnum cupressiforme Hedw. were collected during May 2018. Transfer factors of radionuclides: 226Ra, 232Th, 40Kand 137Cs were calculated with regard to elemental concentrations of radionuclides in soil and moss samples. Analysis was done in order to indicate the different ways of adopting radionuclides by mosses. According to calculated transfer factors and analysis, authors concluded that the soil is dominant source of natural radionuclides and their concentration in moss occurred due to resuspension of soil particles, while artificial 137Cs is present in soil and moss samples as a consequence of atmospheric dry and wet deposition.

Extracts Characterization and In Vitro Evaluation of Potential Immunomodulatory Activities of the Moss Hypnum cupressiforme Hedw.

Recently, there has been an increasing interest in the chemistry and biological potential of mosses, since a large number of biologically active compounds have been found within these species. This study aimed at examining the chemical composition and immunomodulatory potential (antioxidant, antidiabetic, anti-neuroinflammatory/antineurodegenerative, and antitumor activities) of moss Hypnum cupressiforme Hedw. extracts. Corresponding extracts have been obtained applying Soxhlet extractor. The chemical characterization was performed using spectrophotometric assays and liquid chromatography–mass spectrometry (LC-MS). The extracts were analyzed for antioxidant activity and for inhibitory activities on α-glucosidase, α-amylase, acetylcholinesterase, and tyrosinase. Additionally, extracts were tested against four cell lines—MRC-5, BV2, HCT-116, and MDA-MB-231—for antitumor and anti-inflammatory activities. Chemical analysis of extracts revealed the presence of flavonoids, phenolic acids, and triterpenoids. Major compounds identified by LC-MS in H. cupressiforme were kaempferol and five phenolic acids: p-hydroxybenzoic, protocatechuic, p-coumaric, gallic, and caffeic acid. According to biochemical assays the investigated extracts exhibited significant immunomodulatory potential. Significant antiproliferative potential against MDA-MB-231 cells has been observed together with the promising anti-neuroinflammatory application. The obtained data suggest that moss H. cupressiforme is a valuable natural source of biologically active compounds with potential application in the pharmaceutical industry.

Determination of the bioaccumulation of radionuclides and trace elements in mosses using nuclear methods

Τα βρύα αποτελούν ιδανικούς βιοϊχνηθέτες. Δε διαθέτουν ριζικό σύστημα, οπότε όλα τα θρεπτικά συστατικά καθώς και το νερό που χρειάζονται τα παίρνουν κατευθείαν από την υγρή και ξηρή εναπόθεση. Από το 1990 και κάθε 5 χρόνια διεξάγεται μια Ευρωπαϊκή έρευνα στο πλαίσιο του ICP Vegetation Programme σε σχέση με τις συγκεντρώσεις των ιχνοστοιχείων σε φυσικά αναπτυσσόμενα βρύα. Η τελευταία έρευνα πραγματοποιήθηκε το 2015/2016, στην οποία η Ελλάδα συμμετείχε για πρώτη φορά με την ομάδα μας. Στα μέσα του καλοκαιριού του 2016, ενενήντα πέντε (95) δείγματα βρύων Hypnum cupressiforme Hedw. συλλέχθησαν από την περιοχή της Βορείου Ελλάδος. Αφού καθαρίστηκαν, προετοιμάστηκαν για τη μέτρησή τους με τη μέθοδο της Νετρονικής Ενεργοποίησης και της γαμμα- φασματοσκοπίας, για τον προσδιορισμό των συγκεντρώσεων των ιχνοστοιχείων και των ραδιονουκλιδίων αντίστοιχα. Μετρήθηκαν οι συγκεντρώσεις 44 ιχνοστοιχείων (Al, As, Ni, V, Cr, Zn, Fe, Br, Cl, I, Mg, Na, K, Sc, Ti, Mn, Co, Se, Rb, Sb, Au, Th, U, Si, Sr, Ca, Zr, Cs, Ba, La, Ce, Nd, Sm, Gd, Tb, Dy, Tm, Yb, Lu, Hf, Ta, In, Mo, Ag). Για τον προσδιορισμό των πηγών τους, εφαρμόστηκε το μοντέλο PMF5, σύμφωνα με το οποίο αναγνωρίστηκαν 5 πηγές: η σκόνη από το χώμα, το παλαιωμένο θαλασσινό αλάτι, η σκόνη από τον δρόμο, τα εργοστάσια καύσης λιγνίτη και τέλος η πλούσια σε Mn πηγή. Η σκόνη από το χώμα συνεισφέρει στα περισσότερα δείγματα των βρύων σε ποσοστό πάνω από 50%, υποδεικνύοντας ότι η πλειοψηφία των ιχνοστοιχείων μεταφέρεται στα βρύα μέσω της επαναιώρησης του χώματος. Μελετήθηκαν ακόμη οι συγκεντρώσεις των εξής ραδιονουκλιδίων: 7Be, 210Pbuns, 40K, 232Th, 226Ra και 137Cs. Παρατηρούνται μικρές διαφορές στις συγκεντρώσεις τους, ανάλογα με την επιφάνεια συλλογής των βρύων (πάνω από πέτρες, στο έδαφος, σε κλαδιά και κοντά στις ρίζες). Το 7Be παρουσιάζει μεγαλύτερες συγκεντρώσεις στα δείγματα που συλλέχθησαν από το έδαφος από ότι κοντά στις ρίζες, ενώ το 40K, εμφανίζει μεγαλύτερες συγκεντρώσεις στα βρύα που βρίσκονταν κοντά στις ρίζες. Όσον αφορά τα δείγματα χώματος που συλλέχθησαν από τις ίδιες περιοχές με τα βρύα, υπάρχει μια ταύτιση μεταξύ των περιοχών που παρουσιάζουν υψηλές συγκεντρώσεις 137Cs στα βρύα, στα χώματα και αυτών που είχαν χαρακτηριστεί ως οι πιο επιβαρυμένες ακριβώς μετά το ατύχημα στο Τσέρνομπιλ. Τελος, η επαναιώρηση του χώματος είναι η κύρια πηγή της πλειοψηφίας των ιχνοστοιχείων και των ραδιονουκλιδίων στα βρύα.

Early response of biological nitrogen fixation in a mature oak dominated forest to elevated atmospheric CO2 fumigation at BIFoR-FACE

<p>Elevated atmospheric carbon dioxide concentrations are stimulating photosynthesis and carbon sequestration. However, the extent of photosynthetic stimulation in forests under future climates is highly uncertain given that nutrient limitation in soils may constrain the CO<sub>2</sub> fertilization effect. The Birmingham Institute of Forest Research (BIFoR), University of Birmingham established the only global mature temperate deciduous forests Free Air Carbon Dioxide Enrichment (FACE) experiment to study the response of forests to future climates. Fumigation of the forest with ~550 ppm CO<sub>2</sub> started in 2017 and will continue until at least 2026. Soil nutrients cycling including nitrogen transformation in response to elevated atmospheric CO<sub>2</sub> (eCO<sub>2</sub>) fumigation is currently investigated to determine the role of nutrient availability in carbon capture by forests. In this paper, we show preliminary results of the response of asymbiotic biological nitrogen fixation (BNF) in soils and epiphytic bryophytes at BIFoR-FACE following a year of eCO<sub>2</sub> fumigation. It is hypothesized that the demand for available nitrogen by trees will increase under eCO<sub>2</sub> and that competition of roots and soil microbes for available nitrogen will enhance asymbiotic BNF to at least meet microbial metabolic nitrogen demands in the long run. Surface soils (0-5 cm) and epiphytic feather moss (Hypnum cupressiforme) growing on oak tree stems in the FACE site were  collected during the second year of eCO<sub>2</sub> fumigation for the quantification of BNF activity using the <sup>15</sup>N<sub>2</sub> assimilation methods (Saiz et al. 2019). Samples were incubated in 50 mL serum bottles under in situ conditions, followed by the analysis of soil and tissue samples for <sup>15</sup>N signature on an Isotope Ratio Mass Spectrometer for the quantification of BNF activity.</p><p>The BNF activity under eCO<sub>2</sub> were 369% higher than in soils under ambient atmospheric CO<sub>2</sub>. BNF rates associated with feather mosses (Hypnum cupressiforme) did not differ between the eCO<sub>2</sub> and control plots; however, rates under eCO<sub>2</sub> on average were 60% lower than in the control plots. Unlike soils, the moisture of feather mosses correlated significantly (R<sup>2</sup> = 51%) with BNF activity. Among nutrients in soil with implications for BNF activity, the concentrations of Mg, K, Co and Ni were significantly lower in soils under eCO<sub>2</sub> than in the control plots, while in feather moss tissues no differences were observed.  Our preliminary results show that eCO<sub>2</sub> fumigation primed asymbiotic BNF activity in soils. An enhancement of BNF together with the observation of a relatively low nutrient content under eCO<sub>2</sub> points to important changes in nitrogen cycling processes in the early years of CO<sub>2</sub> fumigation. Further detailed studies are underway to fully disentangle controls on nitrogen availability to trees under future climates.</p><p><strong> </strong></p><p><strong>Reference</strong></p><p>Saiz, E, Sgouridis, F, Drifjhout, F & Ullah, S. 2019. Biological nitrogen fixation in peatlands: comparison between acetylene reduction assay and <sup>15</sup>N<sub>2</sub> assimilation methods. Soil Biol. Biochem:131:157-165</p>

Morphological Traits Influence the Uptake Ability of Priority Pollutant Elements by Hypnum cupressiforme and Robinia pseudoacacia Leaves

In this paper, a biomonitoring survey of airborne priority pollutant elements was carried out using leaves of native black locust and moss bags filled with Hypnum cupressiforme. The aims of the work were (i) to evaluate if mosses and leaves provide similar information regarding the accumulation of the elements of environmental concern (As, Be, Cd, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, V, Zn, Tl); (ii) to evaluate if leaf traits are significantly involved in the uptake mechanisms. Hypnum transplants showed elemental contents generally higher than R. pseudoacacia leaves, despite the shorter exposure time. Moss accumulated larger amounts of elements linked to PM and the resuspension of soil dust. Based on the calculation of deposition flux for each element, R. pseudoacacia showed lower values for most elements—except Cr, Mo and Zn—indicating that uptake takes place both by deposition on the leaf surface and absorption via the root. Leaf traits (micromorphology of surface) play an important role in the interception and retention of PM-linked elements. Hypnum transplanted in bags was confirmed to be a powerful bio-accumulator of airborne elements; by contrast, R. pseudoacacia, with a smooth surface and scarce trichomes, showed a limited ability in airborne element retention. Therefore, widely diffused species, well-adapted to anthropized environments, such as black locust, not always can be considered as good biomonitors. The results are discussed in comparison to other vascular plant species used in biomonitoring studies.