Anti-phototoxicity and anti-melanogenesis activities of eelgrass Zostera marina and its phenolic constituents.

The eelgrass Zostera marina L. has several economic roles, from its earlier usage in the insulation industry to protecting the earth from global warming. In this study, we aimed to discover the cosmetic potential of Z. marina. A methanolic extract of Z. marina showed anti-phototoxicity and anti-melanogenesis activity with an IC50 of 17.5 µM, followed by a phytochemical analysis of its phenolic constituents. Ten compounds (1-10) were isolated by several chromatographic techniques and identified by means of nuclear magnetic resonance spectroscopy (NMR) as well as high-resolution mass spectrometry (HR/MS). The identified compounds are caffeic acid (1), 3,4-dihydroxybenzoic acid (protocatechuic acid) (2), luteolin (3), diosmetin (4), 4-coumaroyl-4'-hydroxyl phenyllactic acid (5), rosmarinic acid (6), caffeoyl-4'-hydroxy-phenyllactic acid (isorinic acid) (7), apigenin 7-O-ß-D-glucopyranoside (8), luteolin 7-O-ß-D-glucopyranoside (9), and luteolin 7-sulfate (10). This is the first report to identify compounds 5 and 7 from the family Zosteraceae. The isolated compounds were assessed for their anti-aging abilities and were found to exhibit good anti-phototoxicity and anti-melanogenesis activities by increasing the viability of UVB-irradiated HaCaT cells by 6% to 34% and by inhibiting melanin synthesis in B16 melanoma cells by 44% to 65%.

Phenolic chemistry of the seagrass Zostera marina Linnaeus: First assessment of geographic variability among populations on a broad spatial scale.

The variability of the phenolic content of thirteen populations of Zostera marina L. (six narrow-leaved and seven wide-leaved ecotypes) from different geographical zones, i.e., Baltic Sea, Mediterranean, East and West Atlantic, and East Pacific coasts was evaluated. Depending on the location, three to five phenolic acids and nine to fourteen flavonoids were identified of which an undescribed flavonoid sulfate. The phenolic concentrations of the thirteen populations differ among countries and among sites within countries. However, the same individuals were found almost everywhere. Substantial phenolic concentrations were found at all study sites with the exception of Puck Bay (Baltic Sea). Some geographical differences in the flavonoid content were observed. The highest phenolic diversity was found with specimens from the French Atlantic coast and the lowest with the Northeastern American sample (Cape Cod, MA). Regardless of their leaf width, the content of phenolic compounds was found to be similar and mainly characterized by rosmarinic acid and luteolin 7,3'-disulfate. The results demonstrate that geographic origin influences the phenolic composition of Z. marina primarily in terms of concentration, but not in terms of individual compound identity, despite the large geographic scale and the contrasting climatic and environmental conditions associated with it. This work is the first study to consider the spatial variability of phenolic compounds for a seagrass species on a spatial scale covering four bioregions. This is also the first to compare the phenolic chemistry of the two ecotypes of Z. marina.

Profiling the cell walls of seagrasses from A (Amphibolis) to Z (Zostera).

BACKGROUND: The polyphyletic group of seagrasses shows an evolutionary history from early monocotyledonous land plants to the marine environment. Seagrasses form important coastal ecosystems worldwide and large amounts of seagrass detritus washed on beaches might also be valuable bioeconomical resources. Despite this importance and potential, little is known about adaptation of these angiosperms to the marine environment and their cell walls. RESULTS: We investigated polysaccharide composition of nine seagrass species from the Mediterranean, Red Sea and eastern Indian Ocean. Sequential extraction revealed a similar seagrass cell wall polysaccharide composition to terrestrial angiosperms: arabinogalactans, pectins and different hemicelluloses, especially xylans and/or xyloglucans. However, the pectic fractions were characterized by the monosaccharide apiose, suggesting unusual apiogalacturonans are a common feature of seagrass cell walls. Detailed analyses of four representative species identified differences between organs and species in their constituent monosaccharide composition and lignin content and structure. Rhizomes were richer in glucosyl units compared to leaves and roots. Enhalus had high apiosyl and arabinosyl abundance, while two Australian species of Amphibolis and Posidonia, were characterized by high amounts of xylosyl residues. Interestingly, the latter two species contained appreciable amounts of lignin, especially in roots and rhizomes whereas Zostera and Enhalus were lignin-free. Lignin structure in Amphibolis was characterized by a higher syringyl content compared to that of Posidonia. CONCLUSIONS: Our investigations give a first comprehensive overview on cell wall composition across seagrass families, which will help understanding adaptation to a marine environment in the evolutionary context and evaluating the potential of seagrass in biorefinery incentives.

Seasonal variation of phenolic compounds in Zostera marina (Zosteraceae) from the Baltic Sea.

Seasonal variations of phenolic compounds, in leaves of Zostera marina L. from the Baltic Sea near Kiel/Germany were investigated. Dominant compounds were mono- and disulfated flavonoids and phenylpropanoic acids, in particular luteolin 7,3'-O-disulfate and diosmetin 7-O-sulfate as well as rosmarinic acid, a dimeric phenylpropanoid. All detected sulfated flavones showed similar seasonal trends: there were two significant concentration peaks in June and November. Moreover, two geographically distinct flavonoid chemotypes were identified based on their respective main flavonoid; one chemotype was characterized by the prevalence of luteolin 7,3'-O-disulfate (German Baltic Sea), and the other by the prevalence of diosmetin 7-O-sulfate (Norwegian North Sea). Furthermore, an undescribed tetrameric phenylpropanoid, 7'',8''-didehydrosalvianolic acid B, was isolated and its structure was established by extensive NMR, MS, and CD experiments. This compound inhibited activity of Na+/K+-ATPase in the micro-molar range without any cytotoxic effects against human cancer and normal cells.

Stable Catechol Keto Tautomers in Cytotoxic Heterodimeric Cyclic Diarylheptanoids from the Seagrass Zostera marina.

Two diarylheptanoid heterodimers, zosterabisphenones A (1) and B (2), were isolated from the seagrass Zostera marina. They feature unprecedented catechol keto tautomers, stable because of steric constraints. Their structure elucidation was based on extensive low-temperature NMR studies and ECD and MS data, with the essential aid of DFT prediction of NMR and ECD spectra. Zosterabisphenone B (2) was selectively cytotoxic against the adenocarcinoma colon cancer cell line HCT116 with IC50 3.6 ± 1.1 µM at 48 h.

Degraded Arabinogalactans and Their Binding Properties to Cancer-Associated Human Galectins.

Galectins represent ß-galactoside-binding proteins with numerous functions. Due to their role in tumor progression, human galectins-1, -3 and -7 (Gal-1, -3 and -7) are potential targets for cancer therapy. As plant derived glycans might act as galectin inhibitors, we prepared galactans by partial degradation of plant arabinogalactan-proteins. Besides commercially purchased galectins, we produced Gal-1 and -7 in a cell free system and tested binding capacities of the galectins to the galactans by biolayer-interferometry. Results for commercial and cell-free expressed galectins were comparable confirming functionality of the cell-free produced galectins. Our results revealed that galactans from Echinacea purpurea bind to Gal-1 and -7 with KD values of 1-2 µM and to Gal-3 slightly stronger with KD values between 0.36 and 0.70 µM depending on the sensor type. Galactans from the seagrass Zostera marina with higher branching of the galactan and higher content of uronic acids showed stronger binding to Gal-3 (0.08-0.28 µM) compared to galactan from Echinacea. The results contribute to knowledge on interactions between plant polysaccharides and galectins. Arabinogalactan-proteins have been identified as a new source for production of galactans with possible capability to act as galectin inhibitors.

An Effectual Biosorbent Substance for Removal of Manganese Ions from Aquatic Environment: A Promising Environmental Remediation Study with Activated Coastal Waste of Zostera marina Plant.

In the present research paper, a biosorptive remediation practice for an aqueous medium sample polluted with manganese ions was implemented using the activated coastal waste of the Zostera marina plant. This is the first report in the literature on the utilization of current modified biological waste as a biosorbent substance for the removal of manganese ions from the water environment. The analyses of biosorbent characterization, environmental condition, kinetic, equilibrium, and comparison were performed to introduce the ability of prepared biosorbent for the removal of manganese from the aquatic medium. The biosorbent matter has a rough surface with numerous cavities and cracks and various functional groups for the biosorption of manganese. The environmental conditions significantly affected the manganese purification process, and the optimum working conditions were determined to be biosorbent quantity of 10 mg, pH of 6, manganese concentration of 30 mg L-1, and time of 60 min. The pseudo-second-order model best explained the kinetic data of biosorption operation. The biosorption equilibrium data were best described by the Freundlich isotherm. According to the Langmuir equilibrium model, the maximum purification potency was estimated to be 120.6 mg g-1. The comparison work revealed that the activated coastal waste of the Z. marina plant could be utilized as an effectual and promising biosorbent substance for the remediation of an aquatic environment contaminated with manganese ions.

Arabinogalactan-proteins of Zostera marina L. contain unique glycan structures and provide insight into adaption processes to saline environments.

Seagrasses evolved from monocotyledonous land plants that returned to the marine habitat. This transition was accomplished by substantial changes in cell wall composition, revealing habitat-driven adaption to the new environment. Whether arabinogalactan-proteins (AGPs), important signalling molecules of land plants, are present in seagrass cell walls is of evolutionary and plant development interest. AGPs of Zostera marina L. were isolated and structurally characterised by analytical and bioinformatics methods as well as by ELISA with different anti-AGP antibodies. Calcium-binding capacity of AGPs was studied by isothermal titration calorimetry (ITC) and microscopy. Bioinformatic searches of the Z. marina proteome identified 9 classical AGPs and a large number of chimeric AGPs. The glycan structures exhibit unique features, including a high degree of branching and an unusually high content of terminating 4-O-methyl-glucuronic acid (4-OMe GlcA) residues. Although the common backbone structure of land plant AGPs is conserved in Z. marina, the terminating residues are distinct with high amounts of uronic acids. These differences likely result from the glycan-active enzymes (glycosyltransferases and methyltransferases) and are essential for calcium-binding properties. The role of this polyanionic surface is discussed with regard to adaption to the marine environment.

Heavy metal accumulation and phytoremediation potential by transplants of the seagrass Zostera marina in the polluted bay systems.

Although seagrasses can incorporate heavy metals from the marine environment, few studies have been conducted on heavy metal uptake and phytoremediation potential by seagrass transplants in the heavy metal contaminated sediments. Zostera marina shoots were transplanted in two polluted bay systems on Korean coasts to evaluate the heavy metal contaminations in sediments and the possibility of using Z. marina transplants as a bioindicator and phytoremediation agent. The major concentrated metals in sediments were As, Cu, Fe, and Pb in Jaran Bay, and Cd, Co, Zn, and Hg in Onsan Bay. The Co, Zn, Pb, and Hg concentrations in Z. marina tissues reflected the sediment heavy metal concentrations, and thus the tissue heavy metal concentrations may be used as bio-indicators of the metal contaminations. Since Z. marina transplants accumulated a great amount of heavy metals in their tissues, they may have the phytoremediation potential for the heavy metal contaminated sediments.

Cytotoxicity screening of supercritical fluid extracted seaweeds and phenylpropanoids.

Detached leaves of Posidonia oceanica and Zostera marina creating nuisance at the shores were extracted by means of supercritical CO2 enriched with a co-solvent, compared with that of soxhlet extraction. The extracts and their active compounds which are phenylpropanoids (chicoric, p-coumaric, rosmarinic, benzoic, ferulic and caffeic acids) were screened for cytotoxicity in cancer cell lines including human breast adenocarcinoma (MCF-7, MDA-MB-231, SK-BR-3), human colon adenocarcinoma (HT-29), human cervix adenocarcinoma (HeLa), human prostate adenocarcinoma (PC-3), Mus musculus neuroblastoma (Neuro 2A) cell lines and African green monkey kidney (VERO) as healthy cell line. Supercritical CO2 extracts proved to be more active than soxhlet counterparts. Particularly, Zostera marina extract obtained by supercritical CO2 at 250 bar, 80 °C, 20% co-solvent and a total flow rate of 15 g/min revealed the best IC50 values of 25, 20, 8 µg/ml in neuroblastoma, colon and cervix cancer cell lines. Among the major compounds tested, p-coumaric acid exhibited the highest cytotoxic against colon and cervix cell lines by with IC50 values of 25, 11 µg/ml. As for the effects on healthy cells, the extract was not cytotoxic indicating a selective cytotoxicity. Obtained supercritical CO2 extracts can be utilized as a supplement for preventive purposes.

Bioremediation potential of waste biomaterials originating from coastal Zostera marina L. meadows for polluted aqueous media with industrial effluents.

The studies on novel and efficient biosorbent materials for the promotion of environmental and economic sustainability have been become an up-to-date attempt by scientists for the removal of synthetic dyes from industrial effluents. The biosorbents prepared from biomass based resources are emerging as an alternative promising material for the environmental clean-up because of their low-cost, renewability, eco-friendly, easy availability and so forth characteristics. Hence, for the first time, the biosorption performance of abundantly available natural biowastes originating from coastal Zostera marina L. meadows was explored for the biotreatment of colored industrial effluents in the present study. The biosorption properties of biosorbent for methylene blue as a representative synthetic industrial dye were investigated by means of the operational parameters optimization, kinetic, isotherm, thermodynamic and characterization studies. The operating conditions significantly affected the biosorption process and the optimal values were determined as pH of 8, biosorbent dosage of 10 mg, dye concentration of 15 mg L-1 and contact time of 120 min. Elovich and Freundlich models provided the best fit to the kinetic and isotherm data compared with other applied models, respectively. The negative change in free energy (-10.682 to -8.466 kJ mol-1) indicated a thermodynamically feasible and spontaneous process. The characterization analysis showed that the biosorbent has appropriate chemical and physical properties for the dye biosorption. Thus, the present study displayed that the waste materials originating from coastal Z. marina L. meadows can be applied as a highly efficient as well as cost-effective green generation biosorbent for the clean-up of colored aquatic media.

Anthropogenic nutrient inputs in the NW Iberian Peninsula estuaries determined by nitrogen and carbon isotopic signatures of Zostera noltei seagrass meadows.

Coastal watersheds of the NW Iberian Peninsula have undergone increases in population densities and urbanization over the past decades. However, the effect of altered nutrient inputs to estuarine Zostera noltei ecosystems associated to these anthropogenic pressures remains largely unknown. Eleven Zostera noltei meadows located in the NW Iberian Peninsula were studied to assess the relationship between the anthropogenic pressure over coastal watersheds and the nitrogen and carbon isotopic signatures of Z. noltei meadows. Anthropogenic pressure on the watersheds was estimated from human population data and land cover classes. Carbon and nitrogen stable isotopic analyses were performed on Z. noltei leaves and in the sediments surrounding the rhizospheres. Our results indicate that the N and C isotopic signatures of Z. noltei meadows from the NW Iberian Peninsula reflect the impact of anthropogenic pressures. Nevertheless, these relationships are complex due to several processes acting simultaneously altering the expected isotopic responses.

Metal concentrations in seagrass (Halophila ovalis) tissue and ambient sediment in a highly modified estuarine environment (Sydney estuary, Australia).

Research into sediment-seagrass tissue metal relationships has been undertaken in Sydney estuary due to the recognized role contamination plays in threats to seagrass health. Seagrass (Halophila ovalis) leaf tissue concentrations are elevated in Cu, Pb and Zn and contain the highest reported root Cr concentrations. Seagrass metal concentrations were significantly different between species H. ovalis and Zostera capricorni; between root and leaf tissue; and between sampling locations. Greatest tissue enrichment was for Pb, however metals were not enriched in seagrass relative to surficial sediment. Fine and total sediment metal concentrations were temporally consistent between collection years 2013/15, whereas root tissue metals changed between years and sites and leaf metal contents were temporally inconsistent. Extractable metal concentrations in fine sediment (<62.5 µm) showed moderate significant correlation with root tissue and a weak significant relationship with leaf tissue, whereas total sediment metal showed no such relationships. Management implications are provided.

Low blue carbon storage in eelgrass (Zostera marina) meadows on the Pacific Coast of Canada.

Seagrass habitats provide important ecosystem services, including their ability to take up and store substantial amounts of organic carbon, known as 'blue carbon.' However, the paucity of geospatial and carbon storage information along the Pacific Coast of Canada hinders the inclusion of blue carbon storage data in conservation planning and policy development in coastal habitats. We assessed the carbon storage and accumulation rates in three eelgrass (Zostera marina) meadows in southern Clayoquot Sound on the Pacific Coast of British Columbia. The intertidal and subtidal portions of each meadow were mapped and sampled to estimate eelgrass density, biomass, and carbon, and sediment cores were analyzed to estimate sediment carbon storage and accumulation rates. Aboveground biomass measurements were consistent with estimates for Z. marina in other regions, with average aboveground carbon biomass estimates of 16.78 g C m-2 and 16.25 g C m-2 in the intertidal and subtidal areas, respectively. However, the estimated aboveground to belowground biomass ratio was an order of magnitude higher than for seagrass species in temperate/tropical areas, largely because belowground biomass was up to 10 times lower than for other Z. marina meadows, averaging 6.17 g C m-2 and 5.03 g C m-2 in the intertidal and subtidal zones, respectively. Sediment carbon concentrations did not exceed 1.30%Corg, and carbon accumulation rates ranged from 2.90-39.61 g Corg m-2 yr-1, decreasing with depth and averaging 10.8 ± 5.2 g Corg m-2 yr-1. While sediment carbon stocks were generally higher in the eelgrass meadows relative to non-vegetated reference sites, carbons stocks averaged 1343 ± 482 g Corg m-2, substantially less than global averages. These carbon results confirm that eelgrass does contribute to carbon storage in Clayoquot Sound but at lower rates than identified for more tropical seagrasses. By improving the quantification of site-specific carbon dynamics, eelgrass' role in climate change mitigation and conservation planning can be assessed.

Mechanisms of Antiviral Activity of the Polyphenol Complex from Seagrass of the Zosteraceae Family against Tick-Borne Encephalitis Virus.

Antiviral activity of the polyphenol complex from seagrass of the Zosteraceae family against highly pathogenic strain of the tick-borne encephalitis virus was studied on passaged culture of porcine embryo kidney cells. The antiviral effect of the test compound manifested in a decrease in the infectious titer of the virus and depended on the concentration and application schemes. Polyphenol complex in a concentration of 100 µg/ml suppressed accumulation of the pathogen in the cell culture: pretreatment of the virus reduced its titer by 4 log, pretreatment of cells by 1.4 log, and application of the compound 1 h after cell infection by 2.8 log. Antiviral action of the test compound is determined by direct inactivation of the virus and inhibition of virus replication at the early stage, which attests to potential of this compound in the treatment of tick-borne encephalitis.

Evaluation of four seagrass species as early warning indicators for nitrogen overloading: Implications for eutrophic evaluation and ecosystem management.

Seagrasses are major coastal primary producers and are widely distributed on coasts worldwide. Seagrasses show sensitivity to environmental stress due to their high phenotypic plasticity, and therefore, we evaluated the use of constituent elements in four dominant seagrass species as early warning indicators for nitrogen eutrophication of coastal regions. A meta-analysis was conducted with published data to develop a global benchmark for the selected indicator, which was used to evaluate nitrogen loading at a global scale. A case study at three bays was subsequently conducted to test for local-scale differences in leaf C/N ratios in four seagrasses. Additionally, morphological and physiological metrics of seagrasses were measured from the three locations under varied nitrogen levels to develop further assessment indexes. The benchmark and local study showed that leaf C/N ratios of Zostera marina were sensitive to nitrogen discharge, which could be a highly valuable early warning indicator on a global scale. Moreover, the threshold value of seagrass leaf C/N was determined according to the benchmark to differentiate eutrophic and low nitrogen levels at a local scale. Of the eight phenotypic metrics measured, leaf width, total chlorophyll (a + b), chlorophyll ratio (a/b), and starch in the rhizome were the most effective at discriminating between the three locations and could also be promising indicators for monitoring eutrophication.

Seasonal dynamics of trace elements in sediment and seagrass tissues in the largest Zostera japonica habitat, the Yellow River Estuary, northern China.

Trace element accumulation is an anthropogenic threat to seagrass ecosystems, which in turn may affect the health of humans who depend on these ecosystems. Trace element accumulation in seagrass meadows may vary temporally due to, e.g., seasonal patterns in sediment discharge from upstream areas. In addition, when several trace elements are present in sufficiently high concentrations, the risk of seagrass loss due to the cumulative impact of these trace elements is increased. To assess the seasonal variation and cumulative risk of trace element contamination to seagrass meadows, trace element (As, Cd, Cr, Cu, Pb, Hg, Mn and Zn) levels in surface sediment and seagrass tissues were measured in the largest Chinese Zostera japonica habitat, located in the Yellow River Estuary, at three sites and three seasons (fall, spring and summer) in 2014-2015. In all three seasons, trace element accumulation in the sediment exceeded background levels for Cd and Hg. Cumulative risk to Z. japonica habitat in the Yellow River Estuary, from all trace elements together, was assessed as "moderate" in all three seasons examined. Bioaccumulation of trace elements by seagrass tissues was highly variable between seasons and between above-ground and below-ground biomass. The variation in trace element concentration of seagrass tissues was much higher than the variation in trace element concentration of the sediment. In addition, for trace elements which tended to accumulate more in above-ground biomass than below-ground biomass (Cd and Mn), the ratio of above-ground to below-ground trace element concentration peaked at times corresponding to high water discharge and high sediment loads in the Yellow River Estuary. Overall, our results suggest that trace element accumulation in the sediment may not vary between seasons, but bioaccumulation in seagrass tissues is highly variable and may respond directly to trace elements in the water column.

Macrophytes as bioindicators of heavy metal pollution in estuarine and coastal environments.

The Derwent estuary, in Tasmania (Australia), is highly contaminated with heavy metals with significant levels in both sediments and benthic fauna. However, little is known about metal content in benthic primary producers. We characterized metal content (Arsenic, Cadmium, Copper, Lead, Selenium and Zinc) in twelve species of macrophyte, including red, green, and brown algae, and seagrasses, from the Derwent. The metals, arsenic, copper, lead, and Zinc were detected in all of the macrophytes assessed, but the levels differed between species. Seagrasses accumulated the highest concentrations of all metals; with Zn levels being particularly high in the seagrass Ruppia megacarpa (from the upper Estuary) and Pb was detected in Zostera muelleri (from the middle estuary). Ulva australis was ubiquitous throughout the middle-lower estuary and accumulated Zn in relatively high concentrations. The findings suggest that analysis of multiple species may be necessary for a comprehensive understanding of estuary-wide metal pollution.

The relationship between metal concentrations in seagrass (Zostera capricorni) tissue and ambient fine sediment in modified and near-pristine estuaries (Sydney estuaries, Australia).

Seagrass (Zostera capricorni) tissue metal concentrations in Sydney estuary are some of the highest reported for urban environments. A strong declining metal (As, Co, Cr, Cu, Mn, Pb and Zn) concentration gradient in fine sediment from highly modified to the near-pristine estuarine environments was matched by a concurrent and statistically significant, moderately strong, relationship with declining metal concentrations for leaf, rhizomes and root tissue types. Leaf tissue more consistently expressed metal concentration uptake from sediment. Rhizome tissue contained the highest concentrations for all metals, except Mn, while leaf concentrations were higher for Cr and Zn and roots were more elevated in Cr and Pb. Tissue Cr, Pb and Zn were close to background for reference sites for leaves. Maximum tissue enrichment over background was 25, 19, 47 and 8 times for Cr, Cu, Pb and Zn. Bio-sediment accumulation factors were generally <1 for all metals.

Analytical determination of the reducing and stabilization agents present in different Zostera noltii extracts used for the biosynthesis of gold nanoparticles.

The objective of this work was to ascertain the nature of the components responsible for the reducing and stabilizing properties of Zostera noltii extracts that lead to gold nanoparticle formation using chemical techniques of analysis. In order to achieve this aim, we try the synthesis of AuNPs with three different extracts from plants collected in the Bay of Cádiz (Spain). The n-butanol extract produced the best results. Taking this into account, four fractions were isolated by Sephadex LH-20 column chromatography from this extract and we studied their activity. The chemical study of these fractions led to the isolation of several flavone sulfates and these were identified as the species' responsible for the formation and stabilization of the AuNPs. Flavone sulfates were purified by high performance liquid chromatography and the structures were established by means of spectroscopic methods nuclear magnetic resonance and mass spectroscopy. AuNPs have an average lifetime of about 16weeks. Additionally, the morphology and crystalline phase of the gold nanoparticles were characterized by transmission electron microscopy. The composition of the nanoparticles was evaluated by electron diffraction and energy dispersive X-ray spectroscopy. An 88% of the gold nanoparticles has a diameter in the range 20-35nm, with an average size of 26±2nm.