Protein Tyrosine Phosphatase 1B and α-Glucosidase Inhibitory Phlorotannins from Edible Brown Algae,Ecklonia stolonifera and Eisenia bicyclis

The present work investigates protein tyrosine phosphatase 1B (PTP1B) and the α-glucosidase inhibitory activities of two edible brown algae, Ecklonia stolonifera and Eisenia bicyclis, as well as in their isolated phlorotannins. Since the individual extracts and fractions showed significant inhibitory activities, column chromatography was performed to isolate six phlorotannins, phloroglucinol (1), dioxinodehydroeckol (2), eckol (3), phlorofurofucoeckol-A (4), dieckol (5), and 7-phloroeckol (6). Phlorotannins 3–6 were potent and noncompetitive PTP1B inhibitors with IC₅₀ values ranging from 0.56 to 2.64 μM; 4–6 exhibited the most potent α-glucosidase inhibition with IC₅₀ values ranging from 1.37 to 6.13 μM. Interestingly, 4 and 6 were noncompetitive, while 5 exhibited competitive inhibition in an α-glucosidase assay. E. stolonifera and E. bicyclis as well as their isolated phlorotannins therefore possessed marked PTP1B and α-glucosidase inhibitory activities; this could lead to opportunities in the development of therapeutic agents to control the postprandial blood glucose level and thereby prevent diabetic complications.     

Extracellular secretion of phenolic substances from living brown algae

The chemical structures of ultraviolet (UV)- absorbing substances secreted from the healthy living brown algae, Eisenia bicyclis and Ecklonia kurome, were demonstrated. The living activity of algal cells was critically examined using a confocal laser-scanning microscope after incorporation of fluorescein diacetate (FDA) into the cells. Using thin-layer chromatography (TLC), reversed-phase three-dimensional high-performance liquid chromatography (RP-3D-HPLC) and gas chromatography-mass spectrometry (GC-MS), it was found that the UV-absorbing substances (λ_max 265–270 nm) secreted from the living brown algae mainly consisted of the three monomeric bromophenols, 2,4-dibromophenol, 2,4,6-tribromophenol and dibromo-iodophenol, but not phloroglucinol or phlorotannins. The other minor compounds detected in the secretions were as follows: benzothiazole, fatty acids (14:1, 16:0 and 18:0 acids), franesol, 3-hydroxy-2,4,4-trimethylpentyl 2-methylpropanoate and squalene. Exudation of phloroglucinol and phlorotannins was ascertained to take place only after the cell death of these algae. These results indicate that, whilst the algae are alive, polymeric phlorotannins are strictly kept within the algal body, and only monomeric bromophenols are secreted into the seawater medium.     

Screening test for deodorizing substances from marine algae and identification of phlorotannins as the effective ingredients in Eisenia bicyclis

Aqueous extracts from 33 species of marine algae were assessed for their methyl mercaptan-trapping activity by gas chromatography to search for novel natural oral deodorants. Brown algae belonging to the Laminariales such as Eisenia bicyclis, Ecklonia cava and Ecklonia kurome were found to show remarkable deodorizing action against methyl mercaptan. The effective components in Eisenia bicyclis were identified as a phlorotannin, a group of molecules which are characteristic components of Laminariales. In addition phlorotannins extracted from E. bicyclis were more effective at reducing methyl mercaptan than conventional natural deodorants such as chlorophyll and sodium copper chlorophyllin.Author for Correspondence     

Inhibitory activity of brown algal phlorotannins against glycosidases from the viscera of the turban shell Turbo cornutus

The crude phlorotannins from the brown alga Eisenia bicyclis showed inhibitory activity against 10 of 13 kinds of glycosidases present in the viscera of the turban shell Turbo cornutus. Phloroglucinol and its oligomers – eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol and 8,8′-bieckol (hexamers), and an unidentified tetramer – were isolated from the crude phlorotannins by column and thin-layer chromatography. Phlorofucofuroeckol A, dieckol and 8,8′-bieckol inhibited α-fucosidase, β-galactosidase and β-mannosidase partially purified from T. cornutus, while phloroglucinol, eckol and the unidentified tetramer were weakly active. Dieckol was a competitive inhibitor of α-fucosidase with an inhibition constant (K _i) of 0.12?mM. The amounts of phlorotannins released after the immersion of freshly collected E. bicyclis in seawater or deionized water were estimated by high-performance liquid chromatography. Nearly all the phlorotannins were exuded into the medium following the death of the algae, whereas no phlorotannins were detected in the medium of living algae. These findings indicate that the phlorotannins deter the feeding of marine herbivorous gastropods by inhibiting the glycosidases.     

Antioxidant activities of phlorotannins isolated from Japanese Laminariaceae

The antioxidant activities of brown algal phlorotannins were evaluated using the inhibition of phospholipid peroxidation in the liposome system, and by determining radical scavenging activities against the superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH). Oligomers of phloroglucinol (1,3,5-trihydroxybenzene), eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol and 8,8′-bieckol (hexamers), isolated from the Laminarian brown algae Eisenia bicyclis, Ecklonia cava and Ecklonia kurome, showed potent inhibition of phospholipid peroxidation at 1 μM in the liposome system. The phlorotannins had significant radical scavenging activities against the superoxide anion (50% effective concentration values: 6.5–8.4 μM) and DPPH (50% effective concentration values: 12–26 μM), and were more effective compared to ascorbic acid and α-tocopherol. For the purpose of using phlorotannins as functional food ingredients, the antioxidant activity of a complex of crude phlorotannins and soybean protein was examined. The complex had a pronounced DPPH-radical scavenging activity. These results suggest that phlorotannins are potent anti-inflammatory substances, and that the Laminariaceous brown algae, which are abundant in phlorotannins, may be useful as a new functional foodstuff or supplement with anti-inflammatory activity.     

Inhibitory effects of brown algal phlorotannins on secretory phospholipase A2s, lipoxygenases and cyclooxygenases

The inhibitory effects of brown algal phlorotannins on secretory phospholipase A₂s (sPLA₂s), lipoxygenases (LOXs) and cyclooxygenases (COXs) were determined with an in vitro assay. Oligomers of phloroglucinol; eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol (a hexamer) and 8,8-bieckol (a hexamer) isolated from the brown alga Eisenia bicyclis had pronounced inhibitory effects on sPLA₂ from porcine pancreas and bee venom (IC₅₀ 100–200 M). The phlorotannins inhibited LOX activity more effectively than the well-known LOX inhibitors; resveratrol and epigallocatechin gallate. 8,8-Bieckol, the strongest LOX inhibitor in this study, inhibited soybean LOX and 5-LOX with IC₅₀ values of 38 and 24 M, respectively. Negligible or very weak effects of the phlorotannins on COX-1 and COX-2 were found, except for an inhibitory effect of dieckol on COX-1 (74.7%) and of eckol on COX-2 (43.2%) at 100 M.     

Characterisation of microsatellite loci in the habitat-forming kelp, Ecklonia radiata (Phaeophyceae, Laminariales)

Habitat forming algae play an important role in the ecology of temperate reefs worldwide. Despite this, our understanding of levels of gene flow within and among populations of algae is largely limited to studies on intertidal species; we know comparatively little about important habitat-forming subtidal algae. Here, we develop eight polymorphic microsatellite markers for the characterisation of population genetic diversity and structure in the subtidal kelp, Ecklonia radiata. This large macroalga is the most abundant habitat-forming kelp on the subtidal rocky reefs of temperate Australia and New Zealand where it forms extensive forests that support an astounding diversity of associated taxa.     

Effect of seaweed concentrate fromEcklonia maxima (Osbeck) Papenfuss onMeloidogyne incognita infestation on tomato

Seaweed concentrate (SWC), prepared fromEcklonia maxima, when applied as a soil drench to tomato seedlings, significantly increased plant growth and reduced infestation byMeloidogyne incognita. Foliar applied SWC had little effect on plant growth and increased nematode galling. Ashing SWC reduced the suppressive effect on nematode infestation. In anin vitro experiment, SWC lessened infestation of root-knot nematodes on excised roots of a susceptible cultivar of tomato. Application of the same concentrations of SWC to a nematode-resistant cultivar increased the number of egg masses.     

Antiproliferative activity of sulfated polysaccharide isolated from an enzymatic digest of Ecklonia cava on the U-937 cell line

A sulfated polysaccharide purified from a brown alga Ecklonia cava, having high anticoagulant activity was investigated for its antiproliferative effect on murine colon carcinoma (CT-26), human leukemic monocyte lymphoma (U-937), human promyelocytic leukemia (HL-60), and mouse melanoma (B-16) cell lines. The sulfated polysaccharide isolated and purified from an enzymatic extract of E. cava had a good selective tumor cell growth inhibition effect; its effect on HL-60 and U-937 was especially promising. The IC₅₀ value for the sulfated polysaccharide from E. cava (ECSP) on U-937 was 43.9 μg mL⁻¹. The presence of the sample in the cell culture media stimulated the induction of apoptosis, revealed by nuclear staining with Hoechst 33342. The apoptosis induction was confirmed by the cell cycle analysis, while pronounced sub-G1 phase arrests of 9.5% and 13.8% were also clearly observed when the cells were treated at 15 and 30 μg mL⁻¹ of ECSP in the U-937 cell line, respectively. After a 24-h incubation period, ECSP dose-dependently enhanced the DNA fragmentation on the U-937 cell line as observed in the agarose gel electrophoresis assay. To rule out the action mechanism of ECSP for its anticancer activity, some western blot analyses were conducted with several antibodies (caspase-7, caspase-8, Bax, Bcl-xL, and PARP) and ECSP had a clear effect on the caspase -7 and 8 which cleave protein substrates, including PARP, an inducer of apoptosis responsible for DNA cleavage. Moreover, ECSP controlled the cellular transmembrane molecules like Bax and Bcl-xL. Taken together, the above results demonstrate that the apoptosis for antiproliferative effect of ECSP was clearly induced on U-937 cells.     

Induction of phlorotannins in the brown macroalga<Emphasis Type="Italic"> Ecklonia radiata</Emphasis> (Laminariales,Phaeophyta) in response to simulated herbivory—the first microscopic study

We investigated, for the first time in a microscopic study, the accumulation of phlorotannins as a possible inducible chemical defence against herbivory. Ecklonia radiata (C. Agardh) was mechanically wounded with a cork borer, simulating grazer action, and at intervals of 0, 1, 2, 3, 5, 7 and 9 days after wounding the distribution of phlorotannins and other structural changes were examined by light, fluorescence and electron microscopy. In brown algal cells, phlorotannins (polyphenolic compounds) occur in vesicles known as physodes. In E. radiata, most of the physodes were found in the outer epidermal cell layer, but some were present in the cortical cells and in the innermost medullary cells (sieve elements and hyphal cells). The wound-healing process could be divided into three stages: (i) closing of the medulla by the formation of new medullary cells, (ii) accumulation of phlorotannins (physodes) at the wound area (first in the medullary cells and then in the cortical cells) and in the medullary tissue further away from the wound, and (iii) formation of a new epidermis. The accumulation of phlorotannins started on day 1 and was evident from day 3 on. Our results show structural wound-healing and support wound-sealing functions for phlorotannins and the view that phlorotannins might be considered as inducible anti-herbivory agent in E. radiata. Our results strongly demonstrate the importance of detailed microscopic studies, in addition to chemical analysis, for revealing the localised nature of the brown algal response to wounding.     

Comparative photosynthetic studies of Ecklonia cava (Laminariales,Phaeophyta) bladelets with and without zoosporangial sori

Photosynthetic rates were compared between Ecklonia cava bladelets with and without zoosporangial sori sampled from the subtidal zone (about 5 m deep) in Nabeta Bay, Shimoda, Japan. Photosynthetic rates of bladelets were lower in the sorus portion than in the non-sorus portion on the basis of area, dry weight and chlorophyll a. Respiration rates were higher in the sorus portion than in the non-sorus portion on the basis of area and chlorophyll a, whereas they were almost the same on a dry weight basis. The differences were mainly due to a large difference in dry weight per unit bladelet area between the sorus and the non-sorus portion. Light compensation points were higher in the sorus portion than in the non-sorus portion.     

Japan's nationwide long‐term monitoring survey of seaweed communities known as the “Monitoring Sites 1000”: Ten‐year overview and future perspectives

“Monitoring Sites 1000” – Japan's long‐term monitoring survey was established in 2003, based on the Japanese Government policy for the conservation of biodiversity. Ecological surveys have been conducted on various types of ecosystems at approximately 1000 sites in Japan for 15 years now and are planned to be carried out for 100 years. Since 2008, seaweed communities had been monitored at six sites, featuring the kelp (e.g. Saccharina and Ecklonia; Laminariales) and Sargassum (Fucales) communities in the subarctic and temperate regions of Japan. Annual surveys were carried out during the season when these canopy‐forming seaweeds are most abundant. A non‐destructive quadrat sampling method, with permanent quadrats placed along transects perpendicular to the shoreline, was used to determine species composition, coverage, and vertical distribution of seaweeds at these sites; while destructive sampling was done every 5 years to determine biomass. The occurrence of canopy‐forming species Saccharina japonica (var. japonica) and Ecklonia cava have appeared to be stable at the Muroran (southwestern part of Hokkaido Island) and Shimoda (Pacific coast of middle Honshu Island) sites, respectively; whereas the coverage of Ecklonia radicosa (= Eckloniopsis radicosa) at the Satsuma‐Nagashima site in southern part of Kyushu Island was highly variable until its sudden disappearance from the habitat in 2016. Thalli of E. radicosa lost most of their blades through browsing by herbivorous fish, and thus, this may be one of the causes of the decline. A shift in the community structure related to environmental changes had also been observed at some other sites. Pre‐ and post‐disaster data revealed the impact of the 2011 earthquake and tsunami disasters, including a shift in the vertical distribution of Ecklonia bicyclis (= Eisenia bicyclis) to shallower depths at the Shizugawa site in the Pacific coast of northern Honshu Island, due to seafloor subsidence.     

Management-free techniques for restoration of Eisenia and Ecklonia beds along the central Pacific coast of Japan

Because of the importance of seaweed resources for conservation of thecoastal environment, the provision of habitats of commercially importantspecies and because of use as human food, a study was carried out on bedsof Eisenia bicyclis and Ecklonia cava. This included the mostimportant environmental factors and the development of techniques forrestoration and long-term maintenance of the beds. At a site chosen fordetailed study, the Eisenia and Ecklonia bed was influenced bymany factors, including desiccation, rainfall, low salinity, low light intensity,turbidity, accumulation of suspended sediments, cover by adhering animals,wave and current action and grazing pressure. The distribution of suchseaweed beds is also limited locally by the size and hardness of rock on thebottom, and by fluctuations in the sand level at the base of the reefs.The management-free technique of creating Eisenia and Ecklonia beds is based on an understanding of the factors limiting the localdistribution of the beds. Conditions for seaweed growth can then beartificially enhanced by coastal engineering. Suggestions to enhance Eisenia and Ecklonia bed restoration using management-freetechniques include raising the bottom, elevating substrata above the sandybottom, and providing substrata of a shape suitable for the attachment ofkelp.     

Relation between size and age of holdfasts of Ecklonia stolonifera Okamura (Laminariales,Phaeophyta) in northern Honshu,Japan

Ecklonia stolonifera is distributed along the coast facing the Sea of Japan. The size of various parts of the shoot (blade length and width and stipe length and diameter) and the age were determined at Ooma, Aomori Prefecture. The smaller the holdfast, the higher the percentage of one-year-old shoots. Holdfasts 10 cm in diameter seemed to be three years old, whereas holdfasts 40 cm in diameter seemed to be five or more years old. Zoosporangial sori were observed on blades three or more years old. Ecklonia stolonifera holdfast diameter expands only vegetatively by stoloniferous rhizoids. Zoospores, formed on shoots three or more years old, serve for the formation of new populations.     

Tissue culture of Ecklonia radiata (Phaeophyceae,Laminariales): effects on growth of light,organic carbon source and vitamins

The effects of light quality and irradiance, and supply of organic carbon and vitamins on the growth of two forms of Ecklonia radiata in tissue culture were examined. A callus of unpigmented cells developed over the cut surface of newly excised explants of stipe. This growth was best in the dark but stopped after 10 weeks. Pigmented, mainly filamentous clumps of cells developed from explants after several weeks in culture. These required light for growth, with growth being enhanced by increasing photon flux density up to 30 μmol photon m^-2 s^-1, with the active spectral component being red light (> 600 nm). The addition to the medium of a range of organic carbon sources or vitamins did not stimulate growth of either culture type in the dark. author for correspondence     

Chemotaxonomic studies onArachniodes (dryopteridaceae) III. Phloroglucinol derivatives of putative hybrids

Phenetic variation found in JapaneseArachniodes was compared with phloroglucinol composition. The differences in this chemotaxonomic feature were not found very useful in defining the taxa inArachniodes. The phloroglucinol compositions of the putative hybrids were determined and compared with those of the presumed parent species. It was concluded that, in general, the hybrids combine the phloroglucinols found in their parent species. Hybridization among the Japanese species ofArachniodes should, however, be confirmed by cytological investigations. Five species of Kalimantan (Bornean) ferns were also studied for their phloroglucinol contents.Arachniodes tripinnata was compared with Philippine materials of the same species with the result that they differed to some extent. In addition, four species belonging to Tectarioideae were examined:Heterogonium is devoid of phloroglucinols, andPleocnemia irregularis contains a great deal of a “yellow compound” of unknown structure.     

Settlement of seaweeds on coastal structures

Coastal structures are constructed principally to protect the coast line. However, these structures also can act as artificial substrata for seaweeds. In particular, armor blocks, such as tetrapods, prove to be good algal substrata. Our field observations on the vegetation and standing crop of seaweed communities on armor blocks led us to the following conclusions: 1) Ecklonia cavacommunities grew on tetrapods that had been placed to coincide with the maturation period of E. cava. The communities have been maintained for more than four years. 2) The standing crop of seaweeds on an offshore breakwater composed of tetrapods placed seven years before was almost the same as that found under natural conditions. 3) Variations in roughened surfaces (pebbles or scores and grooves 3 or 46 mm wide) did not affect the growth of Sargassum spp. However, a greater number of Ecklonia stolonifera plants were observed attached to roughened surfaces compared to smooth surfaces.     

Predicting understorey structure from the presence and composition of canopies: an assembly rule for marine algae

Assembly rules provide a useful framework for predicting patterns of community assembly under defined environmental conditions. Habitat created by canopy-forming algae (such as kelps) provides a promising system for identifying assembly rules because canopies typically have a large and predictable influence on understorey communities. Across >1,000 km of subtidal South Australian coastline, we identified natural associations between assemblages of understorey algae and (1) monospecific canopies of Ecklonia radiata, (2) canopies comprised of E. radiata mixed with Fucales (Cystophora spp. and Sargassum spp.), and (3) gaps among canopies of algae. We were able to recreate these associations with experimental tests that quantified the assembly of understorey algae among these three habitat types. We propose the assembly rule that understorey communities on subtidal rocky coast in South Australia will be (1) monopolised by encrusting coralline algae beneath monospecific canopies of E. radiata, (2) comprised of encrusting corallines, encrusting non-corallines, and sparse covers of articulated corallines, beneath mixed E. radiata-Fucales canopies, and (3) comprised of extensive covers of articulated corallines and filamentous turfs, as well as sparse covers of foliose algae and juvenile canopy-formers, within gaps. Consistencies between natural patterns and experimental effects demonstrate how algal canopies can act as a filter to limit the subsets of species from the locally available pool that are able to assemble beneath them. Moreover, the subsets of species that assemble to subtidal rocky substrata in South Australia appear to be predictable, given knowledge of the presence and composition of canopies incorporating E. radiata.     

Potential of Candida utilis to ferment Ecklonia cava by-product for enhanced anti-methicillin-resistant Staphylococcus aureus (MRSA) activity

In order to utilize the by-product of Ecklonia cava (the remaining biomass of E. cava), microbial fermentation which may result in the production of bioactive compounds using the by-product was applied in this study. The fermentation broth of E. cava by-product fermented with Candida utilis showed enhanced antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and food-borne pathogenic bacteria compared to that of control. To perform a more detailed investigation on the antibacterial activity, the broth was extracted with methanol and further fractionated with organic solvents. After 1 day of fermentation, the ethyl acetate (EtOAc) fraction exhibited the highest anti-MRSA activity with minimum inhibitory concentration ranging from 64 to 256 μg mL^?1, suggesting that the fermentation of E. cava by-product with C. utilis could enhance antibacterial activity against MRSA. In addition, high-performance liquid chromatography analysis revealed that dieckol, eckol, eckstolonol, and triphlorethol-A contents in the EtOAc-soluble extract increased significantly. The anti-MRSA activity of E. cava by-product most probably originated from phlorotannins, and the fermentation of C. utilis may have stimulated the breakdown of phlorotannins or have increased the efficiency in extracting phlorotannins.     

Effect of seaweed concentrate on the establishment and yield of greenhouse tomato plants

Seaweed concentrate prepared fromEcklonia maxima (Osbeck) Papenfuss, when applied as a soil drench, significantly improved the growth of tomato seedlings. Application as a foliar spray had no effect on young plants. In a second experiment SWC-treated plants exhibited early fruit ripening and a total fruit fresh weight increase of 17%. The number of harvested fruit were improved by about 10%. In this instance foliar applied SWC was more beneficial than SWC applied to the soil. The significance of these findings is discussed.author for correspondence