Sulfated polysaccharides from brown seaweeds Saccharina japonica and Undaria pinnatifida: isolation, structural characteristics, and antitumor activity

During the last decade brown seaweeds attracted much attention as a source of polysaccharides, namely laminarans, alginic acids, and sulfated polysaccharides—fucoidans, with various structures and biological activities.In this study, sulfated polysaccharides were isolated from brown seaweeds Saccharina japonica (formerly named Laminaria) and Undaria pinnatifida and their antitumor activity was tested against human breast cancer T-47D and melanoma SK-MEL-28 cell lines.The sulfated polysaccharide form S. japonica was highly branched partially acetylated sulfated galactofucan, built up of (1→3)-α-l-fucose residues. The sulfated polysaccharide from U. pinnatifida was partially acetylated highly sulfated galactofucan consisting of (1→3)- or (1→3);(1→4)-α-l-fucose residues.Fucoidans from S. japonica and U. pinnatifida distinctly inhibited proliferation and colony formation in both breast cancer and melanoma cell lines in a dose-dependent manner. These results indicated that the use of sulfated polysaccharides from brown seaweeds S. japonica and U. pinnatifida might be a potential approach for cancer treatment.     

Antimutagenicity of a suberin extract from Quercus suber cork

The possible protective effect of a suberin extract from Quercus suber cork on acridine orange (AO)-, ofloxacin- and UV radiation-induced mutagenicity (bleaching activity) in Euglena gracilis was examined. To our knowledge, the present results are the first attempt to analyse suberin in relation to mutagenicity of some chemicals. Suberin exhibits a significant dose-dependent protective effect against AO-induced mutagenicity and the concentration of 500 μg/ml completely eliminates the Euglena-bleaching activity of AO. The mutagenicity of ofloxacin is also significantly reduced in the presence of suberin (125, 250 and 500 μg/ml). However, the moderate protective effect of suberin on UV radiation-induced mutagenicity was observed only at concentrations 500 and 1000 μg/ml. Our data shows that suberin extract from Q. suber cork possess antimutagenic properties and can be included in the group of natural antimutagens acting in a desmutagenic manner.     

Sulfated polysaccharides from marine green algae Ulva conglobata and their anticoagulant activity

Sulfated polysaccharides from the green algae Ulva conglobata were isolated and prepared by extraction in hot water, precipitation with ethanol and purification by ion-exchange and size-exclusion column chromatography. The characterizations of the sulfated polysaccharides were defined, and containing 23.04–35.20% sulfate ester groups, 10.82–14.91% uronic acid and 3.82–4.51% protein. Gas chromatography analysis shows that the sulfated polysaccharides from Ulva conglobata are mainly consisted of rhamnose with variable contents of glucose and fucose, trace amounts of xylose, glactose and mannose. The anticoagulant properties of the sulfated polysaccharides were compared with those of heparin by studying the activated partial thromboplastin time using normal human plasma. The sulfated polysaccharide from Ulva conglobata collected in Qingdao, China is the most potent among the sulfated polysaccharides tested. The mechanism of anticoagulant activity mediated by the sulfated polysaccharides is due to the direct inhibition of thrombin and the potentiation of heparin cofactor II.     

Evaluation of the mutagenic properties of the coccolithophorePleurochrysis carterae (Haptophyceae) as a potential human food supplement

The mutagenicity of the algaPleurochrysis carterae for use as human food was tested by the Ames method with the modification of pre-incubation, by usingSalmonella typhimurium TA98, TA100, TA1535, TA1537 andEscherichia coli WP2uvrA. The freeze-dried powder ofP. carterae was not mutagenic to any strain either with or without S9 mix. In view of the absence of adverse effects ofP. carterae in this mutagenicity study, it is suggested thatP. carterae is safe for human consumption as a human food supplement.Author for correspondence     

Preparation,structural characterization and <Emphasis Type="Italic">in vitro</Emphasis> antitumor activity of <Emphasis Type="Italic">kappa</Emphasis>-carrageenan oligosaccharide fraction from <Emphasis Type="Italic">Kappaphycus striatum</Emphasis>

Oligosaccharides were prepared through mild hydrochloric acid hydrolysis of kappa-carrageenan from Kappaphycus striatum to compare the antitumor activity with carrageenan polysaccharides. Oligosaccharide fractions were isolated by gel permeation chromatography and the structure of fraction 1 (F1) was studied by using negative-ion electrospray ionization-mass spectrometry (ESI-MS), and ¹H and ¹³C-NMR spectrometry. The in vitro antitumor effects in three human neoplastic cell lines (KB, BGC, and Hela) of polysaccharides and F1 were investigated. The bioassay results showed that F1 exhibited relatively higher antitumor activity against the three cancer cells than polysaccharides.     

Extraction, characterization of Astragalus polysaccharides and its immune modulating activities in rats with gastric cancer

One major polysaccharide fractions, glucose, were isolated from the polysaccharides extract of Astragalus (AP), a valuable traditional Chinese medicine, using thin-layer chromatography (TLC) and Sephadex G-100 chromatography. HPLC and IR methods were used for a qualitative and quantitative determination of from polysaccharides of Astragalus. The HPLC method was validated for linearity, precision and accuracy. The results indicated that polysaccharides of Astragalus is an α-(1 → 4)-d-glucan with α-(1 → 6)-linked branches attached to the O-6 of branch points. Bioactivity tests showed that polysaccharides of Astragalus is active for spleen lymphocytes proliferation. The polysaccharides also presented anti-inflammatory activities. These data together suggest that polysaccharides of Astragalus presents significant immune modulating activity, thus supporting the popular use of the polysaccharides in the treatment of gastric cancer diseases.     

Adulteration identification of some fungal polysaccharides with SEM, XRD, IR and optical rotation: A primary approach

Edible mushrooms have been consumed in Asian countries for healthy functions historically with polysaccharides, which have been manufactured as ending products, as one of the major bioactive components. Driven by profits, some inglorious manufacturers conducted adulteration using cheap carbohydrates. The extracted polysaccharides from Agaricus blazei Murill, Ganoderma lucidum and Lentinus edodes were chosen as products and maltodextrin, carboxymethyl cellulose (CMC), water soluble corn starch and guar gum as adulterants for adulteration investigation. Scanning electron microscope (SEM) observation implied regular granular shapes of fungal polysaccharides different significantly from the adulterants. Infrared spectra revealed α- and β-anomeric types of polysaccharides and carbohydrates. X-ray diffractometry (XRD) suggested different diffraction patterns of polysaccharides from adulterant carbohydrates, the former were amorphous while the later somewhat crystalline of different crystallinity. Chiral properties of polysaccharides and carbohydrates showed big differences in specific rotation with good correlation between the content of “pure” fungal polysaccharides and adulterants.     

Cloning, heterologous gene expression and biochemical characterization of the alpha-1,3-glucanase from the filamentous fungus Penicillium purpurogenum

There has been much recent interest in α-1,3-glucanases (mutanases) as they have the potential to be used in the treatment of dental caries. Mutanases have been reported in a number of bacteria, yeast and fungi but remain a relatively uncharacterised family of enzymes. In this study we heterologously expressed the mutanase gene from the filamentous fungus Penicillium purpurogenum to enable further characterization of its enzymatic activity. The mutanase cDNA was cloned and expressed in the methylotrophic yeast Pichia pastoris. The molecular mass of the secreted protein was about 102 kDa. The recombinant enzyme hydrolyzed mutan with a specific activity of 3.9 U/mg of protein. The recombinant enzyme was specific for mutan and could not cleave a variety of other polysaccharides demonstrating a specificity for α-1,3-glucosidic linkages. The pH and temperature optima were pH 4.6 and 45 °C, respectively. Synthetic compounds were also tested as substrates to assess whether the P. purpurogenum mutanase has an exo- or endo-type mechanism of hydrolysis. The results suggest an endo-hydrolytic mode of action. The type of mechanism was confirmed since mutanase activity was not suppressed in the presence of inhibitors of exo-type enzymes.     

Assessment of the carcinogenic potential of a proposed food coloring additive,laccaic acid,using short-term assays

Laccaic acid is a red colored natural dye produced by the insect Laccifer lacca or Coccus lacca. It is obtained in large amounts as a by-product of the shellac industry and has been considered for general use as a food coloring agent. Laccaic acid is found to have no mutagenic activity as assessed by two short-term assays: the Salmonella/microsome mutagenicity test, and the ØX fidelity assay. However, laccaic acid did inhibit metabolic cooperation in Chinese hamster V79 cells. These results suggest that laccaic acid should be tested in animals with particular emphasis on in vivo models for tumor promotion.     

Mutagenic activity of the glutathione S-transferase substrate 1-chloro-2,4-dinitrobenzene (CDNB) in the Salmonella mutagenicity assay

The mutagenicity of the commonly used glutathione S-transferase substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) was investigated in the Salmonella mutagenicity assay. CDNB induced a concentration-dependent mutagenic response in Salmonella typhimurium strain TA98. Incorporation of an activation system derived from Aroclor 1254-induced rats did not influence mutagenic response. Under the same conditions DCNB failed to display mutagenic activity. The mutagenic activity of CDNB was attenuated in bacterial strains under-expressing nitroreductase or O-acetylase activity but, in contrast, it was exaggerated in an O-acetylase over-expressing strain. It is inferred that CDNB exhibits a mutagenic response following reduction of the nitro-group to the hydroxylamine, which is further acetylated to form the acetoxy derivative that presumably breaks down spontaneously to generate the nitrenium ion, the likely ultimate mutagen.     

Mannan transglycosylase: a novel enzyme activity in cell walls of higher plants

Mannan transglycosylase is a novel cell wall enzyme activity acting on mannan-based plant polysaccharides in primary cell walls of monocotyledons and dicotyledons. The enzyme activity was detected by its ability to transfer galactoglucomannan (GGM) polysaccharides to tritium-labelled GGM-derived oligosaccharides generating tritium-labelled GGM polysaccharides. Mannan transglycosylase was found in a range of plant species and tissues. High levels of the enzyme activity were present in flowers of some kiwifruit (Actinidia) species and in ripe tomato (Solanum lycopersicum L.) fruit. Low levels were detected in mature green tomato fruit and activity increased during tomato fruit ripening up to the red ripe stage. Essentially all activity was found in the tomato skin and outermost 2 mm of tissue. Mannan transglycosylase activity in tomato skin and outer pericarp is specific for mannan-based plant polysaccharides, including GGM, galactomannan, glucomannan and mannan. The exact structural requirements for valid acceptors remain to be defined. Nevertheless, a mannose residue at the second position of the sugar chain and the absence of a galactose substituent on the fourth residue (counting from the non-reducing end) appear to be minimal requirements. Mannan-based polysaccharides in the plant cell wall may have a role analogous to that of xyloglucans, introducing flexibility and forming growth-restraining networks with cellulose. Thus mannan transglycosylase and xyloglucan endotransglycosylase, the only other known transglycosylase activity in plant cell walls, may both be involved in remodelling and refining the cellulose framework in developmental processes throughout the life of a plant.Abbreviations EBM Endo--mannanase - GGM galactoglucomannan - GGMO Galactoglucomannan-derived oligosaccharide - G₂M₅ Di-galactosyl mannopentaitol - M₂–M₅ Mannobiitol to mannopentaitol oligosaccharides - SK+OP Skin plus outer pericarp - XET Xyloglucan endotransglucosylase - XG Xyloglucan     

Improvement of lipid profile and antioxidant of hypercholesterolemic albino rats by polysaccharides extracted from the green alga Ulva lactuca Linnaeus

Sulfated polysaccharides from Ulva lactuca were extracted in hot water and precipitated by ethanol then orally gavaged to rats fed on a hypercholesterolemic diet for 21 days to evaluate the antihypercholesterolemic and antioxidant actions. Atorvastatine Ca (Lipitor) was used as a reference drug. The intragastric administration of U. lactuca extract to hypercholesterolemic rats caused significant decrease of serum total lipids, triglycerides, total cholesterol, LDL-cholesterol and vLDL-cholesterol levels. Whereas, HDL-cholesterol concentration was markedly increased by 180%. Aqueous extract showed a significant ameliorative action on elevated atherogenic index, creatine kinase and lactate dehydrogenase activities of hypercholesterolemic group. Furthermore, serum activities of transaminases and alkaline phosphatase were also improved. High fat diet intake caused a highly significantly elevated serum urea, creatinine concentration. These effects were reversed by oral administration of U. lactuca extract. Sulfates polysaccharides extract of U. lactuca ameliorate hepatic enzymatic (catalase, glutathione peroxidase and superoxide dismutase), non-enzymatic (reduced glutathione & total thiol) antioxidant defenses and thiobarbituric acid reactive substances. In conclusion, the tested U. lactuca polysaccharides extract has potent hypocholesterolemic and antioxidant effects in experimentally-induced hypercholesterolemic animal model.     

Antioxidant acitivity in vitro and in vivo of the capsule polysaccharides from Streptococcus equi subsp. zooepidemicus

Crude capsule polysaccharides (CCP) were prepared from the culture of Streptococcus equi subsp. zooepidemicus C55129 and were partially purified through an anion-exchange column chromatography to afford partially purified capsule polysaccharides (PCP). The main component of CCP and PCP was hyaluronic acid. In vitro antioxidant assay, the capsule polysaccharides showed strong inhibition of lipid peroxidation and hydroxyl radical scavenging activity and moderate 1,1-diphenyl-2-picryldydrazyl radical scavenging activity. In addition, CCP exhibited much stronger reductive power than PCP. For antioxidant testing in vivo, CCP and PCP were orally administrated over a period of 15 days in a d-galactose induced aged mice model. As results, administration of capsule polysaccharides inhibited significantly the formation of malondialdehyde in mice livers and serums and raised the activities of antioxidant enzymes and total antioxidant capacity in a dose-dependent manner. However, the antioxidant activity of CCP was lower than that of PCP. The results suggest that the capsule polysaccharides from Streptococcus equi subsp. zooepidemicus C55129 have direct and potent antioxidant activities.     

Formation of antigenic extracellular polysaccharides by selected strains ofMucor spp.,Rhizopus spp.,Rhizomucor spp.,Absidia corymbifera andSyncephalastrum racemosum

In this study, polyclonal IgG antibodies raised against extracellular polysaccharides (EPS) ofMucor racemosus were characterised as almost specific for moulds belonging to the order of Mucorales. Cross-reactivity in the ELISA could be observed only towards the yeastPichia membranaefaciens. EPS were isolated from various cultures ofM. hiemalis growing on six different carbon sources and two nitrogen sources, with ratios varying from 0.13 to 0.44 relative to the amount of biomass. Other strains includingMucor spp.,Rhizopus spp.,Rhizomucor spp.,Absidia corymbifera andSyncephalastrum racemosum also excreted EPS, with ratios varying from 0.05 to 0.23. In all cases, the excreted EPS had similar antigenic properties as determined by ELISA. No enzymatic degradation of the antigenic parts of the polysaccharides could be observed upon prolonged incubation. Considering that all tested strains formed similar amounts of antigenic EPS there might be scope for the specific detection of biomass of Mucoralean moulds using ELISA techniques for example in food.     

The use of the Salmonella BA9 forward mutation assay in sediment quality assessment: mutagenicity of freshly deposited sediments of the River Elbe

The aim of the present study was to investigate whether bacterial mutagenicity assays can be applied in sediment quality assessment. The arabinose-sensitive forward mutation strain Salmonella typhimurium BA 9 was added to freshly deposited sediments of the River Elbe, collected during March, April, and May 1994. Up to twelve locations were sampled each month and mutagenicity was determined employing the solid phase and sequentially prepared toluene- and methanol-extracts of the sediments. Mutagenicity was detected at all sites within the solid sediments without addition of S9-mix; however, the addition of mammalian enzymes (S9) enhanced the mutagenic effect. In contrast, mutagenicity of toluene extracts containing the lipophilic fraction of the sediment samples was higher in the absence of S9-mix; peaks of mutagenic activity in these samples were observed at Dessau (mouth of the River Mulde) and close to the city of Hamburg (Bunthaus). Similar results were obtained with methanolic extracts of the sediments, although the effects were usually lower in comparison to the corresponding toluene extracts. These results show that the mutagenicity assays are capable of assessing water/sediment contamination and reveal that the mutagenicity detected in sediments reflects local industrial activities as well as hydrologic conditions.     

Inhibition of doxorubicin-induced mutagenicity by Baccharis dracunculifolia

Baccharis dracunculifolia DC (Asteraceae), a native plant from Brazil, have been used as an antipyretic, stomachic and health tonic in Brazil. The objective of the present study was to investigate the potential mutagenic effect of B. dracunculifolia ethyl acetate extract (Bd-EAE) and its influence on the mutagenicity induced by the chemotherapeutic agent doxorubicin (DXR) using the rat bone marrow and peripheral blood micronucleus test. Wistar rats were divided into 10 treatment groups. Five groups received DXR (90 mg/kg body weight, b.w., intraperitoneally) to induce mutagenicity and three of these groups received a single oral dose of Bd-EAE at a concentration of 6, 12 or 24 mg/kg b.w. prior to DXR administration. A vehicle-treated control group and Bd-EAE control groups were also included. The results showed that Bd-EAE itself was not mutagenic, in the rat micronucleus assay. In animals treated with Bd-EAE and DXR, the number of MNPCEs was significantly decreased compared to animals receiving DXR alone. HPLC analysis of the extract obtained permitted the identification of the following phenolic compounds: caffeic acid, p-coumaric acid, aromadendrin-4′O-methyl ether, 3-prenyl-p-coumaric acid (drupanin), 3,5-diprenyl-p-coumaric acid (artepillin C) and baccharin. The putative antioxidant activity or the interference of one or more of the active compounds of Bd-EAE with mutagenic metabolic pathways may explain its effect on DXR mutagenicity.     

Mutagenicity,sister chromatid exchange inducibility andin vitro cell transforming ability of particulates from Athens air

Airborne particulates were collected over a period of twelve months by the use of Hi-Vol samplers in the basin of Athens, Greece. N-Hexane extracts were tested in a battery ofin vitro tests for their ability to induce mutation in bacteria as well as mutation, sister chromatid exchange and morphological transformation in cultured mammalian cells. Positive results were found for mutagenicity withSalmonella strain TA98 in the Ames assay, for sister chromatid exchange induction in CHO cells and for transformation in BALB/c 3T3 cells in culture. They also showed weak non-doserelated induction of ouabain resistance in BALB/c 3T3 cells. The contribution of oxidizing and nitrating agents found in the Athens atmosphere, together with sunlight UV irradiation in the formation of direct acting mutagens and potential carcinogens from ambient polycyclic aromatic hydrocarbons, is suggested.Abbreviations FCS fetal calf serum - FPG fluorescent-plus-Giemsa technique - oua^R ouabain resistant - PAH polycyclic aromatic hydrocarbon - SCE sister chromatid exchange - TSP total suspended particulate     

Evaluation of the antimutagenic potential of chitosan oligosaccharide: Rec, Ames and Umu tests

Two types of chitosan oligosaccharides (COS), COS I (1-kDa < MW < 3-kDa) and COS II (3-kDa < MW < 5-kDa), were tested for antimutagenic activities against chemical mutagens using Umu gene expression, Ames, and Bacillus subtilis Rec mutagenicity tests. At the highest chitosan oligosaccharide dose (1 mg) tested, mutagenic activity of indirect-acting mutagen was inhibited by 50% in the Umu gene expression system and in the Ames test. Chitosan oligosaccharide (0.01, 0.1 and 1 mg) also suppressed 4-nitroquinoline-N-oxide (NQO)-induced mutagenicity in the B. subtilis Rec^– assay.     

Direct addition of cultures of tester bacteria into semi-permeable membrane devices (SPMDs) as a modified procedure for preliminary detection of mutagenic pollution of the marine environment by use of microbiological mutagenicity assays

Mutagenic pollution of the natural environment, including marine waters, is a very serious ecological problem. However, since chemical mutagens usually occur and act at low concentrations, their detection and identification is technically difficult, laborious and time-consuming. Therefore, preliminary detection of mutagenic pollution is commonly based on biological mutagenicity assays. On the other hand, triolein-containing semi-permeable membrane devices (SPMDs) provide a method for concentration of hydrophobic organic contaminants, including a large fraction of the mutagens. Combinations of SPMDs with microbiological toxicity and mutagenicity assays have already been described, but only SPMD-derived extracts, prepared with various organic solvents, were tested in such a way to date. We found that the presence of these solvents could interfere with the Vibrio harveyi bioluminescence-based mutagenicity assay. Moreover, preparation of the extracts from SPMD takes usually at least 48 h. Here, we propose a modified procedure, based on direct addition of tester bacteria cultures into SPMD. We found that this procedure is significantly (at least two times) more rapid and several times more sensitive than that based on testing the extracts. This optimization is presented in this report. Moreover, we have performed preliminary studies on samples of marine waters. Positive results (i.e. detection of mutagenic activity) were obtained when test samples came from a region known to be highly contaminated by industrial pollution, while negative results were observed in the case of samples from a region supposed to be of low risk for mutagenic pollution.