Structural Characteristics and Anticancer Activity of Fucoidan from the Brown Alga Sargassum mcclurei

Three different fucoidan fractions were isolated and purified from the brown alga, Sargassum mcclurei. The SmF1 and SmF2 fucoidans are sulfated heteropolysaccharides that contain fucose, galactose, mannose, xylose and glucose. The SmF3 fucoidan is highly sulfated (35%) galactofucan, and the main chain of the polysaccharide contains a →3)-α-l-Fucp(2,4SO₃⁻)-(1→3)-α-l-Fucp(2,4SO₃⁻)-(1→ motif with 1,4-linked 3-sulfated α-l-Fucp inserts and 6-linked galactose on reducing end. Possible branching points include the 1,2,6- or 1,3,6-linked galactose and/or 1,3,4-linked fucose residues that could be glycosylated with terminal β-d-Galp residues or chains of alternating sulfated 1,3-linked α-l-Fucp and 1,4-linked β-d-Galp residues, which have been identified in galactofucans for the first time. Both α-l-Fucp and β-d-Galp residues are sulfated at C-2 and/or C-4 (and some C-6 of β-d-Galp) and potentially the C-3 of terminal β-d-Galp, 1,4-linked β-d-Galp and 1,4-linked α-l-Fucp residues. All fucoidans fractions were less cytotoxic and displayed colony formation inhibition in colon cancer DLD-1 cells. Therefore, these fucoidan fractions are potential antitumor agents.     

Structure of the Cell-Wall-Associated Polysaccharides from the Deep-Sea Marine Bacterium Devosia submarina KMM 9415T

Two cell-wall-associated polysaccharides were isolated and purified from the deep-sea marine bacterium Devosia submarina KMM 9415^T, purified by ultracentrifugation and enzymatic treatment, separated by chromatographic techniques, and studied by sugar analyses and NMR spectroscopy. The first polysaccharide with a molecular weight of about 20.7 kDa was found to contain d-arabinose, and the following structure of its disaccharide repeating unit was established: →2)-α-d-Araf-(1→5)-α-d-Araf-(1→. The second polysaccharide was shown to consist of d-galactose and a rare component of bacterial glycans-d-xylulose: →3)-α-d-Galp-(1→3)-β-d-Xluf-(1→.     

Structural and Immunological Activity Characterization of a Polysaccharide Isolated from Meretrix meretrix Linnaeus

Polysaccharides from marine clams perform various biological activities, whereas information on structure is scarce. Here, a water-soluble polysaccharide MMPX-B2 was isolated from Meretrix meretrix Linnaeus. The proposed structure was deduced through characterization and its immunological activity was investigated. MMPX-B2 consisted of d-glucose and d-galctose residues at a molar ratio of 3.51:1.00. The average molecular weight of MMPX-B2 was 510 kDa. This polysaccharide possessed a main chain of (1→4)-linked-α-d-glucopyranosyl residues, partially substituted at the C-6 position by a few terminal β-d-galactose residues or branched chains consisting of (1→3)-linked β-d-galactose residues. Preliminary immunological tests in vitro showed that MMPX-B2 could stimulate the murine macrophages to release various cytokines, and the structure-activity relationship was then established. The present study demonstrated the potential immunological activity of MMPX-B2, and provided references for studying the active ingredients in M. meretrix.     

Structural Studies of the Lipopolysaccharide from the Fish Pathogen Aeromonas veronii Strain Bs19, Serotype O16

Chemical analyses, mass spectrometry, and NMR spectroscopy were applied to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas veronii strain Bs19, serotype O16. ESI-MS revealed that the most abundant LPS glycoforms have tetra-acylated or hexa-acylated lipid A species, consisting of a bisphosphorylated GlcN disaccharide with an AraN residue as a non-stoichiometric substituent, and a core oligosaccharide composed of Hep₅Hex₃HexN₁Kdo₁P₁. Sugar and methylation analysis together with 1D and 2D ¹H and ¹³C NMR spectroscopy were the main methods used, and revealed that the O-specific polysaccharide (OPS) of A. veronii Bs19 was built up of tetrasaccharide repeating units with the structure: →4)-α-d-Quip3NAc-(1→3)-α-l-Rhap-(1→4)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→. This composition was confirmed by mass spectrometry. The charge-deconvoluted ESI FT-ICR MS recorded for the LPS preparations identified mass peaks of SR- and R-form LPS species, that differed by Δm = 698.27 u, a value corresponding to the calculated molecular mass of one OPS repeating unit (6dHexNAc6dHexHexHexNAc-H₂O). Moreover, unspecific fragmentation spectra confirmed the sequence of the sugar residues in the OPS and allowed to assume that the elucidated structure also represented the biological repeating unit.     

Production,Characterization, and Antioxidant Activity of Fucoxanthin from the Marine Diatom Odontella aurita

The production, characterization, and antioxidant capacity of the carotenoid fucoxanthin from the marine diatom Odontella aurita were investigated. The results showed that low light and nitrogen-replete culture medium enhanced the biosynthesis of fucoxanthin. The maximum biomass concentration of 6.36 g L⁻¹ and maximum fucoxanthin concentration of 18.47 mg g⁻¹ were obtained in cultures grown in a bubble column photobioreactor (Ø 3.0 cm inner diameter), resulting in a fucoxanthin volumetric productivity of 7.96 mg L⁻¹ day⁻¹. A slight reduction in biomass production was observed in the scaling up of O. aurita culture in a flat plate photobioreactor, yet yielded a comparable fucoxanthin volumetric productivity. A rapid method was developed for extraction and purification of fucoxanthin. The purified fucoxanthin was identified as all-trans-fucoxanthin, which exhibited strong antioxidant properties, with the effective concentration for 50% scavenging (EC₅₀) of 1,1-dihpenyl-2-picrylhydrazyl (DPPH) radical and 2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical being 0.14 and 0.03 mg mL⁻¹, respectively. Our results suggested that O. aurita can be a natural source of fucoxanthin for human health and nutrition.     

Extraction,Isolation, Structural Characterization and Anti-Tumor Properties of an Apigalacturonan-Rich Polysaccharide from the Sea Grass Zostera caespitosa Miki

An apigalacturonan (AGA)-rich polysaccharide, ZCMP, was isolated from the sea grass Zostera caespitosa Miki. The depolymerized fragments derived from ZCMP were obtained by either acidic degradation or pectinase degradation, and their structures were characterized by electrospray ionization collision-induced-dissociation mass spectrometry (ESI-CID-MS²) and nuclear magnetic resonance (NMR) spectroscopy. The average molecular weight of ZCMP was 77.2 kD and it consisted of galacturonic acid (GalA), apiosefuranose (Api), galactose (Gal), rhamnose (Rha), arabinose (Ara), xylose (Xyl), and mannose (Man), at a molar ratio of 51.4꞉15.5꞉6.0꞉11.8꞉4.2꞉4.4꞉4.2. There were two regions of AGA (70%) and rhamnogalacturonan-I (RG-Ι, 30%) in ZCMP. AGA was composed of an α-1,4-d-galactopyranosyluronan backbone mainly substituted at the O-3 position by single Api residues. RG-Ι possessed a backbone of repeating disaccharide units of →4GalAα1,2Rhaα1→, with a few α-l-arabinose and β-d-galactose residues as side chains. The anti-angiogenesis assay showed that ZCMP inhibited the migratory activity of human umbilical vein endothelial cell (HUVECs), with no influence on endothelial cells growth. ZCMP also promoted macrophage phagocytosis. These findings of the present study demonstrated the potential anti-tumor activity of ZCMP through anti-angiogenic and immunoregulatory pathways.     

Synthesis and Bioactivity of Ancorinoside B,a Marine Diglycosyl Tetramic Acid

The sponge metabolite ancorinoside B was prepared for the first time in 16 steps and 4% yield. It features a β-d-galactopyranosyl-(1→4)-β-d-glucuronic acid tethered to a d-aspartic acid-derived tetramic acid. Key steps were the synthesis of a fully protected d-lactose derived thioglycoside, its attachment to a C₂₀-aldehyde spacer, functionalization of the latter with a terminal N-(β-ketoacyl)-d-aspartate, and a basic Dieckmann cyclization to close the pyrrolidin-2,4-dione ring with concomitant global deprotection. Ancorinoside B exhibited multiple biological effects of medicinal interest. It inhibited the secretion of the cancer metastasis-relevant matrix metalloproteinases MMP-2 and MMP-9, and also the growth of Staphylococcus aureus biofilms by ca 87% when applied at concentrations as low as 0.5 µg/mL. This concentration is far below its MIC of ca 67 µg/mL and thus unlikely to induce bacterial resistance. It also led to a 67% dispersion of preformed S. aureus biofilms when applied at a concentration of ca 2 µg/mL. Ancorinoside B might thus be an interesting candidate for the control of the general hospital, catheter, or joint protheses infections.     

Hydrolysis of Fucoidan by Fucoidanase Isolated from the Marine Bacterium,Formosa algae

Intracellular fucoidanase was isolated from the marine bacterium, Formosa algae strain KMM 3553. The first appearance of fucoidan enzymatic hydrolysis products in a cell-free extract was detected after 4 h of bacterial growth, and maximal fucoidanase activity was observed after 12 h of growth. The fucoidanase displayed maximal activity in a wide range of pH values, from 6.5 to 9.1. The presence of Mg²⁺, Ca²⁺ and Ba²⁺ cations strongly activated the enzyme; however, Cu²⁺ and Zn²⁺ cations had inhibitory effects on the enzymatic activity. The enzymatic activity of fucoidanase was considerably reduced after prolonged (about 60 min) incubation of the enzyme solution at 45 °C. The fucoidanase catalyzed the hydrolysis of fucoidans from Fucus evanescens and Fucus vesiculosus, but not from Saccharina cichorioides. The fucoidanase also did not hydrolyze carrageenan. Desulfated fucoidan from F. evanescens was hydrolysed very weakly in contrast to deacetylated fucoidan, which was hydrolysed more actively compared to the native fucoidan from F. evanescens. Analysis of the structure of the enzymatic products showed that the marine bacteria, F. algae, synthesized an α-l-fucanase with an endo-type action that is specific for 1→4-bonds in a polysaccharide molecule built up of alternating three- and four-linked α-l-fucopyranose residues sulfated mainly at position 2.     

New α-Glucosidase Inhibitory Triterpenic Acid from Marine Macro Green Alga Codium dwarkense Boergs

The marine ecosystem has been a key resource for secondary metabolites with promising biological roles. In the current study, bioassay-guided phytochemical investigations were carried out to assess the presence of enzyme inhibitory chemical constituents from the methanolic extract of marine green alga—Codium dwarkense. The bioactive fractions were further subjected to chromatographic separations, which resulted in the isolation of a new triterpenic acid; dwarkenoic acid (1) and the known sterols; androst-5-en-3β-ol (2), stigmasta-5,25-dien-3β,7α-diol (3), ergosta-5,25-dien-3β-ol (4), 7-hydroxystigmasta-4,25-dien-3-one-7-O-β-d-fucopyranoside (5), 7-hydroxystigmasta-4,25-dien-3-one (6), and stigmasta-5,25-dien-3β-ol (7). The structure elucidation of the new compound was carried out by combined mass spectrometry and 1D (¹H and ¹³C) and 2D (HSQC, HMBC, COSY, and NOESY) NMR spectroscopic data. The sub-fractions and pure constituents were assayed for enzymatic inhibition of alpha-glucosidase. Compound 1 showed significant inhibition at all concentrations. Compounds 2, 3, 5, and 7 exhibited a dose-dependent response, whereas compounds 4–6 showed moderate inhibition. Utilizing such marine-derived biological resources could lead to drug discoveries related to anti-diabetics.     

Synthesis of the Oligosaccharides Related to Branching Sites of Fucosylated Chondroitin Sulfates from Sea Cucumbers

Natural anionic polysaccharides fucosylated chondroitin sulfates (FCS) from sea cucumbers attract great attention nowadays due to their ability to influence various biological processes, such as blood coagulation, thrombosis, angiogenesis, inflammation, bacterial and viral adhesion. To determine pharmacophore fragments in FCS we have started systematic synthesis of oligosaccharides with well-defined structure related to various fragments of these polysaccharides. In this communication, the synthesis of non-sulfated and selectively O-sulfated di- and trisaccharides structurally related to branching sites of FCS is described. The target compounds are built up of propyl β-d-glucuronic acid residue bearing at O-3 α-l-fucosyl or α-l-fucosyl-(1→3)-α-l-fucosyl substituents. O-Sulfation pattern in the fucose units of the synthetic targets was selected according to the known to date holothurian FCS structures. Stereospecific α-glycoside bond formation was achieved using 2-O-benzyl-3,4-di-O-chloroacetyl-α-l-fucosyl trichloroacetimidate as a donor. Stereochemical outcome of the glycosylation was explained by the remote participation of the chloroacetyl groups with the formation of the stabilized glycosyl cations, which could be attacked by the glycosyl acceptor only from the α-side. The experimental results were in good agreement with the SCF/MP2 calculated energies of such participation. The synthesized oligosaccharides are regarded as model compounds for the determination of a structure-activity relationship in FCS.     

Activation of Dormant Secondary Metabolite Production by Introducing Neomycin Resistance into the Deep-Sea Fungus,Aspergillus versicolor ZBY-3

A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(d-Pro-d-Phe) (1), cyclo(d-Tyr-d-Pro) (2), phenethyl 5-oxo-l-prolinate (3), cyclo(l-Ile-l-Pro) (4), cyclo(l-Leu-l-Pro) (5) and 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1–6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 μg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal metabolic pathways. This approach could be applied to elicit the metabolic potentials of other fungal isolates to discover new compounds from cryptic secondary metabolites.     

Three New Cytotoxic Polyhydroxysteroidal Glycosides from Starfish Craspidaster hesperus

Three new polyhydroxysteroidal glycosides, hesperuside A (1), B (2), and C (3), as well as a known novaeguinoside A (4), were isolated from the ethanol extract of starfish Craspidaster hesperus collected from the South China Sea. Their structures were elucidated by extensive spectroscopic methods and chemical evidence. The compounds 1–3 present unprecedented carbohydrate chain 3-O-methyl-β-d-galactopyranose, which differ from each other in the side chains. These compounds exhibited cytotoxicity against human tumor cells BEL-7402, MOLT-4, and A-549 in vitro.     

Molecular Mechanism of Anti-Inflammatory Activities of a Novel Sulfated Galactofucan from Saccharina japonica

Seaweed of Saccharina japonica is the most abundantly cultured brown seaweed in the world, and has been consumed in the food industry due to its nutrition and the unique properties of its polysaccharides. In this study, fucoidan (LJNF3), purified from S. japonica, was found to be a novel sulfated galactofucan, with the monosaccharide of only fucose and galactose in a ratio of 79.22:20.78, and with an 11.36% content of sulfate groups. NMR spectroscopy showed that LJNF3 consists of (1→3)-α-l-fucopyranosyl-4-SO₃ residues and (1→6)-β-d-galactopyranose units. The molecular mechanism of the anti-inflammatory effect in RAW264.7 demonstrated that LJNF3 reduced the production of nitric oxide (NO), and down-regulated the expression of MAPK (including p38, ENK and JNK) and NF-κB (including p65 and IKKα/IKKβ) signaling pathways. In a zebrafish experiment assay, LJNF3 showed a significantly protective effect, by reducing the cell death rate, inhibiting NO to 59.43%, and decreasing about 40% of reactive oxygen species. This study indicated that LJNF3, which only consisted of fucose and galactose, had the potential to be developed in the biomedical, food and cosmetic industries.     

Structure of Fucoidan from Brown Seaweed Turbinaria ornata as Studied by Electrospray Ionization Mass Spectrometry (ESIMS) and Small Angle X-ray Scattering (SAXS) Techniques

The purpose of this study is to elucidate both the chemical and conformational structure of an unfractionated fucoidan extracted from brown seaweed Turbinaria ornata collected at Nha-trang bay, Vietnam. Electrospray ionization mass spectrometry (ESI-MS) was used for determining the chemical structure and small angle X-ray scattering (SAXS) provided conformational of the structure at the molecular level. The results showed that the fucoidan has a sulfate content of 25.6% and is mainly composed of fucose and galactose residues (Fuc:Gal ≈ 3:1). ESIMS analysis suggested that the fucoidan has a backbone of 3-linked α-l-Fucp residues with branches, →4)-Galp(1→ at C-4 of the fucan chain. Sulfate groups are attached mostly at C-2 and sometimes at C-4 of both fucose and galactose residues. A molecular model of the fucoidan was built based on obtained chemical structure and scattering curves estimated from molecular model and observed SAXS measurement were fitted. The results indicated that fucoidan under study has a rod-like bulky chain conformation.     

Three New Asperentin Derivatives from the Algicolous Fungus Aspergillus sp. F00785

Three new asperentin-type compounds, 6-O-α-d-ribosylasperentin (1) and 6-O-α-d-ribosyl-8-O-methylasperentin (2) and 5-hydroxyl-6-O-methylasperentin (3), along with asperentin (4) and its known analogues (5–9), were isolated from a halotolerant Aspergillus sp. strain {"type":"entrez-nucleotide","attrs":{"text":"F00785","term_id":"707638","term_text":"F00785"}}F00785, an endotrophic fungus from marine alga. Their structures were determined using extensive NMR and HRESIMS spectroscopic analysis, including the X-ray crystallographic data for the assignment of the absolute configurations of compound 9. Compound 4 exhibited highly potent inhibitory activity against crop pathogens, Colletotrichum gleosporioides Penz. and Colletotrichum gleosporioides (Penz.) Sacc.     

Isolation and Structure Elucidation of GM4-Type Gangliosides from the Okinawan Starfish Protoreaster nodosus

Three new ganglioside molecular species, termed PNG-1, PNG-2A, and PNG-2B were isolated from pyloric caeca of the starfish Protoreaster nodosus. Their structures were elucidated using a combination of spectroscopic and chemical methods, and characterized as 1-O-[8-O-methyl-N-acetyl-α-neuraminosyl-(2→3)-β-galactopyranosyl]-ceramide for PNG-1, 1-O-[β-galactofuranosyl-(1→3)-α-galactopyranosyl-(1→4)-8-O-methyl-N-acetyl-α-neuraminosyl-(2→3)-β-galactopyranosyl]-ceramide for PNG-2A, and 1-O-[β-galactofuranosyl-(1→3)-α-galactopyranosyl-(1→9)-N-acetyl-α-neuraminosyl-(2→3)-β-galactopyranosyl]-ceramide for PNG-2B. PNG-2A and PNG-2B represent the first GM4 elongation products in nature.     

Structural and Immunochemical Studies of the Lipopolysaccharide from the Fish Pathogen,Aeromonas bestiarum Strain K296, Serotype O18

Chemical analyses and mass spectrometry were used to study the structure of the lipopolysaccharide (LPS) isolated from Aeromonas bestiarum strain K296, serotype O18. ESI-MS revealed that the most abundant A. bestiarum LPS glycoforms have a hexa-acylated or tetra-acylated lipid A with conserved architecture of the backbone, consisting of a 1,4′-bisphosphorylated β-(1→6)-linked d-GlcN disaccharide with an AraN residue as a non-stoichiometric substituent and a core oligosaccharide composed of Kdo₁Hep₆Hex₁HexN₁P₁. 1D and 2D NMR spectroscopy revealed that the O-specific polysaccharide (OPS) of A. bestiarum K296 consists of a branched tetrasaccharide repeating unit containing two 6-deoxy-l-talose (6dTalp), one Manp and one GalpNAc residues; thus, it is similar to that of the OPS of A. hydrophila AH-3 (serotype O34) in both the sugar composition and the glycosylation pattern. Moreover, 3-substituted 6dTalp was 2-O-acetylated and additional O-acetyl groups were identified at O-2 and O-4 (or O-3) positions of the terminal 6dTalp. Western blots with polyclonal rabbit sera showed that serotypes O18 and O34 share some epitopes in the LPS. The very weak reaction of the anti-O34 serum with the O-deacylated LPS of A. bestiarum K296 might have been due to the different O-acetylation pattern of the terminal 6dTalp. The latter suggestion was further confirmed by NMR.     

Antiviral Sulfoquinovosyldiacylglycerols (SQDGs) from the Brazilian Brown Seaweed Sargassum vulgare

Total lipids from the Brazilian brown seaweed Sargassum vulgare were extracted with chloroform/methanol 2:1 and 1:2 (v/v) at room temperature. After performing Folch partition of the crude lipid extract, the lipids recovered from the Folch lower layer were fractionated on a silica gel column eluted with chloroform, acetone and methanol. The fraction eluted with methanol, presented a strong orcinol-positive band characteristic of the presence of sulfatides when examined by TLC. This fraction was then purified by two successive silica gel column chromatography giving rise to fractions F4I86 and F4II90 that exhibited strong activity against herpes simplex virus type 1 and 2. The chemical structures present in both fractions were elucidated by ESI-MS and ¹H/¹³C NMR analysis HSQC fingerprints based on their tandem–MS behavior as sulfoquinovosildiacylglycerols (SQDGs). The main SQDG present in both fractions and responsible for the anti-herpes activity observed was identified as 1,2-di-O-palmitoyl-3-O-(6-sulfo-α-d-quinovopyranosyl)-glycerol.     

Antioxidant Activities of Hydrolysates of Arca Subcrenata Prepared with Three Proteases

In order to get products with antioxidant activity from Arca subcrenata Lischke, the optimal hydrolase and hydrolysis conditions were investigated in the paper. Three proteases (neutrase, alcalase and papain) were applied to hydrolyze the homogenate of A. subcrenata. An orthogonal design was used to optimize hydrolysis conditions, and the pH-stat methods was used to determine the degree of hydrolysis. Viewed from the angle of reducing power, such as scavenging activities against α,α-diphenyl-β-picrylhydrazyl (DPPH) radical and hydrogen peroxide, the antioxidant activities of the alcalase hydrolysate (AH) were superior to neutrase hydrolysate (NH) and papain hydrolysate (PH), and its EC₅₀ values in DPPH radical and hydrogen peroxide scavenging effect were 6.23 mg/ml and 19.09 mg/ml, respectively. Moreover, compared with products hydrolyzed by neutrase and papain, the molecular mass of AH was lower and its content of amino acid of peptides was higher. Therefore, alcalase was selected as the optimal enzyme to produce active ingredients since its hydrolysate exhibited the best antioxidant activity among them and possessed large amount of potential active peptides.