Chemical composition of lipopolysaccharides isolated from various endophytic nitrogen-fixing bacteria of the genus Herbaspirillum.

Bacteria from the genus Herbaspirillum are endophytes responsible for nitrogen fixation in gramineous plants of economic importance such as maize, sugarcane, sorghum, rice, and wheat. Some species are known to produce plant growth substances. In contrast, Herbaspirillum rubrisubalbicans strains are known to be mild plant pathogens. The molecular communication between the plant and the microbes might involve lipopolysaccharides present in the outer membrane of these gram-negative bacteria. Phenol-water extraction was used to obtain lipopolysaccharides from 7 strains of Herbaspirillum seropedicae (SmR1, Z67, Z78, ZA95, and M2) and H. rubrisubalbicans (M1 and M4). The electrophoretic profiles and chemical composition of the lipopolysaccharides obtained in the phenol and aqueous extracts were shown herein.

A 3-O-methylated mannogalactan from Pleurotus pulmonarius: structure and antinociceptive effect.

A polysaccharide (Mw 2.39x10(4)g/mol) was extracted with cold water from the basidiomycete Pleurotus pulmonarius, and its antinociceptive and anti-inflammatory properties were evaluated. It was a mannogalactan (MG), whose structure was characterized using mono- and two-dimensional NMR spectroscopy, methylation analysis, and a controlled Smith degradation. It had a main chain of (1-->6)-linked alpha-D-galactopyranosyl and 3-O-methyl-alpha-D-galactopyranosyl units, both of which are partially substituted at O-2 by beta-D-mannopyranosyl non-reducing ends. The MG was tested for its effects on the acetic acid-induced writhing reaction in mice, a typical model for inflammatory pain, causing a marked and dose-dependent inhibition of the nociceptive response, with ID50 of 16.2 (14.7-17.7)mg/kg and inhibition of 93+/-3% at a dose of 30mg/kg. An inflammatory response was not inhibited.

Safe therapeutics of murine melanoma model using a novel antineoplasic, the partially methylated mannogalactan from Pleurotus eryngii.

A heteropolysaccharide was isolated by cold aqueous extraction from edible mushroom Pleurotus eryngii ("King Oyster") basidiocarps and its biological properties were evaluated. Structural assignments were carried out using mono- and bidimensional NMR spectroscopy, monosaccharide composition, and methylation analyses. A mannogalactan having a main chain of (1→6)-linked α-d-galactopyranosyl and 3-O-methyl-α-d-galactopyranosyl residues, both partially substituted at OH-2 by ß-d-Manp (MG-Pe) single-unit was found. Biological effects of mannogalactan from P. eryngii (MG-Pe) were tested against murine melanoma cells. MG-Pe was non-cytotoxic, but reduced in vitro melanoma cells invasion. Also, 50mg/kg MG-Pe administration to melanoma-bearing C57BL/6 mice up to 10days decreased in 60% the tumor volume compared to control. Additionally, no changes were observed when biochemical profile, complete blood cells count (CBC), organs, and body weight were analyzed. Mg-Pe was shown to be a promising anti-melanoma molecule capable of switching melanoma cells to a non-invasive phenotype with no toxicity to melanoma-bearing mice.

Anticoagulant and antithrombotic activities of a chemically sulfated galactoglucomannan obtained from the lichen Cladonia ibitipocae.

A galactoglucomannan (GGM), isolated from the lichen Cladonia ibitipocae, consisted of a (1-->6)-linked main chain of alpha-mannopyranose units, substituted by alpha- and beta-D-galacto (alpha- and beta-D-Galp)-, beta-D-gluco (beta-D-Glcp)- and alpha-D-mannopyranosyl (alpha-D-Manp) groups, and was sulfated giving a sulfated polysaccharide (GGM-SO4) with 42.2% sulfate corresponding to a degree of substitution of 1.29. NMR studies indicated that after sulfation, the OH-6 groups of galactopyranosyl and mannopyranosyl units were preferentially substituted. GGM-SO4 was investigated in terms of its in vitro anticoagulant and in vivo antithrombotic properties. Those of the former were evaluated by its activated partial thromboplastin (APTT) and thrombin time (TT), using pooled normal human plasma, and compared with that of 140 USP units mg(-1) for a porcine intestinal mucosa heparin. Anticoagulant activity was detected in GGM-SO4, but not in GGM. The in vivo antithrombotic properties of GGM-SO4 were evaluated using a stasis thrombosis model in Wistar rats, intravenous administration of 2 mg kg(-1) body weight totally inhibiting thrombus formation. It caused dose-dependent increases in tail transection bleeding time. The results obtained showed that this sulfated polysaccharides is a promising anticoagulant and antithrombotic agent.

Characterization of lyso-galactolipids, C-2 and C-3 O-acyl trigalactosylglycerol isomers, obtained from the lichenized fungus Dictyonema glabratum.

A mixture of two lyso isomers of a galactolipid was obtained from Dictyonema glabratum. Aqueous hydrolysis gave rise to galactose and glycerol in a 3:1 molar ratio. ESI-MS spectroscopy gave, in the positive-ion mode, a pseudomolecular ion at m/z 839 and daughter ions with m/z 677, 600, 515 and 353, suggesting three galactosyl units linked to a glycerol moiety, substituted by one O-acyl group. 1D and 2D NMR experiments were used to characterize the glycolipid, and HMQC examination showed three anomeric signals, corresponding to two alpha-Galp and one beta-Galp residue liked to glycerol. The glycolipid structure was shown to be O-alpha-D-Galp-(1-->6)-O-alpha-D-Galp-(1-->6)-O-beta-D-Galp-(1<-->1)-2- and -3-monoacyl-D-glycerol, the latter structures not having been previously found in nature. The fatty acid composition was determined by GC-MS of derived methyl esters: that of palmitic acid C(16:0) was the most abundant, although the presence of C(12:0), C(14:0), C(16:1) and C(18:0) esters was observed.

Galactomannoglucans of lichenized fungi of Cladonia spp.: significance as chemotypes.

The chemical structures of the glucans, galactoglucomannans and galactomannoglucans of two species of the Cladonia, section Cocciferae, Cladonia miniata and Cladonia salmonea, were determined and compared. alpha-D-Glucans of the nigeran type were isolated from both species, in common with all Cladonia spp., along with galactoglucomannans containing (1-->6)-linked main-chains of alpha-D-Manp units substituted by structurally different and typical side-chains. Isolated were previously unreported galactomannoglucans, with (1-->3)-linked main-chains of beta-D-Glcp units, substituted at O-2,6 by side-chains. These consisted of beta-D-Galf, 6-O-substituted beta-D-Galf and 2-O-, 4-O-, 6-O- and 2, 3-di-O-substituted alpha-D-Manp units. According to (13)C NMR spectroscopy, a similar galactomannoglucan was isolated from the Cladonia spp. Cladonia signata, Cladonia crispatula, Cladonia penicillata, Cladonia imperialis, Cladonia clathrata, Cladonia connexa, Cladonia substellata and Cladonia ibitipocae. Its presence could also contribute to the classic taxonomy of lichenized fungi.

Comparative studies of the polysaccharides isolated from lichenized fungi of the genus Cladonia: significance as chemotypes.

Beta-D-glucans of the laminaran type were prepared from 15 Cladonia spp., Cladonia bellidiflora, Cladonia boryi, Cladonia clathrata, Cladonia connexa, Cladonia crispatula, Cladonia furcata, Cladonia gracilis, Cladonia ibitipocae, Cladonia imperialis, Cladonia miniata, Cladonia penicillata, Cladonia salmonea, Cladonia signata, Cladonia substellata and Cladonia uncialis. They were extracted with 10% aqueous KOH at 100 degrees C, giving polysaccharides with varying yields and proportions of mannose, galactose and glucose. Their aqueous solutions were freeze-thawed giving precipitates of mixed alpha-glucan (nigeran) and beta-glucans, which were isolated and suspended in aqueous 0.5% KOH at 50 degrees C, which preferentially dissolved the beta-glucan. In the case of the C. uncialis product, it was subjected to methylation analysis, which gave rise to 2,4,6-tri-O-methylglucitol acetate only, corresponding to (1-->3)-linkages. Its specific rotation (+4 degrees ) and one- and two-dimensional nuclear magnetic resonance (NMR) spectra were consistent with beta-linkages. 13C and (1)H-1 signals were observed, respectively, at delta 102.8 (C-1), 86.0 (C-3), 76.2 (C-5), 72.6 (C-2), 68.3 (C-4) and 60.7 (C-6), and 4.55 (H-1), 3.31 (H-2), 3.49 (H-3), 3.27 (H-4), 3.27 (H-5), 3.48 (H-6) and 3.72 (H-6'). Similar (13)C-NMR spectra were obtained from the glucans from the other 14 Cladonia spp. The beta-D-glucans of the laminaran type seems to be present in all Cladonia spp. being significant for chemotyping since it was observed in every species studied.

An unusual glucomannan from Tornabenia intricata.

Mannose-containing polysaccharides of 18 lichen species were prepared via successive alkaline extraction, precipitation with Fehling solution and fractional precipitation with Cetavlon. Products from Fehling and Cetavlon precipitation, the latter at pH 8.5 in the presence of borax, were structurally similar, except with those of Usnea sp., U. meridionalis, Parmotrema araucaria and Evernia prunastri, which were mixtures and initially provided precipitates at pH 7 due to the presence of carboxyl groups. With one exception, glucosyl units were detected in all preparations, but possibly arose from glucan contaminants of the galactomannans. Tornabenia intricata, however, did not contain galactose, and a glucomannan was isolated. It consisted of two components with M(r)s of ca 0.85 x 10(5) and ca 1.1 x 10(5) and whose 13C NMR spectra were identical. The overall preparation contained a (1-->6)-linked alpha-D-Manp main-chain substituted at 0-2 mainly with side chains of alpha-D-Manp with smaller amounts of alpha-D-Glcp, alpha-D-Glcp-(1-->2)-[alpha-D-Manp-(1-->4)]-alpha-D-Manp, and possibly alpha-D-Manp-(1-->2)-[alpha-D-Manp-(1-->4)]-alpha-D-Manp+ ++.

Unusual carbohydrates from the lichen, Parmotrema cetratum.

The lichen Parmotrema cetratum contains traces of the unusual threitol and unexpected volemitol, along with galactose (2%). Present is a complex containing a lightly branched beta-glucan containing (1-->3) and (1-->4)-linkages in a 25:47 molar ratio chemically linked to a galactomannan with structural features common in other lichens. A glucogalactomannan with a small proportion of Glc rho side chains was also characterized.

Gum heteropolysaccharide and free reducing mono- and oligosaccharides of Anadenanthera colubrina.

The gum from Anadenanthera colubrina consists mainly of a complex high-arabinose heteropolysaccharide with a (1-->3)-linked beta-D-Galp main-chain and many different side-chains. These contain beta-D-Galp-[(1-->6)-beta-D-Galp]m-(1-->6)-, substituted in turn at O-3 by alpha-L-Araf-[(1-->3)-alpha-L-Araf-]0-2. Also present are (1) main-chain units substituted at O-4 and O-6 by alpha-L-Araf units, (2) side-chains of Rhap-(1-->4)-beta-D-GlcpA-(1-->6)-beta-Galp-groups, (3) alpha-L-Arap non-reducing end-units linked (1-->6) to D-Galp, and (4) beta-Araf and beta-Arap structures. For the first time, a plant gum exudate was found to contain in the natural state, reducing low M(r) carbohydrates. These were rhamnose (0.6%), arabinose (4.7%), mannose (0.1%), galactose (0.8%) and many oligosaccharides (0.6%; 11 with different RFs, with the majority containing arabinose). They were all mixtures with the exception of alpha-Rhap-(1-->4)-beta-D-GlcpA-(1-->6)-alpha beta-Gal and an incompletely identified hexasaccharide, probably having alpha-L-Araf-(1-->4)-beta-D-Galp- and -alpha-L-Araf-(1-->3)-beta-D-Galp- structures. The mono- and oligosaccharides do not appear to arise via in situ autohydrolysis of the gum.

Similarity of monosaccharide, oligosaccharide and polysaccharide structures in gum exudate of Anacardium occidentale.

The gum exudate from the Brazilian cashew-nut tree (Anacardium occidentale) contained traces of the reducing sugars, rhamnose (0.005%), arabinose (0.03%), mannose (0.007%), galactose (0.03%), glucose (0.02%), beta-D-Galp-(1-->6)-alpha beta-D-Gal (0.05%), alpha-L-Rhap-(1-->4)-alpha beta-D-GlcA (0.008%) and alpha-L-Rhap-(1-->4)-beta-D-GlcpA-(1-->6)-beta-D-Galp-(1-->6 )-alpha beta-D-Gal (0.008%). Rhamnose, arabinose, glucose and the three oligosaccharides are components of the side-chains of the gum polysaccharide, which has a main chain of (1-->3)-linked beta-D-Galp units. The structure of this polysaccharide was determined and found to differ from that previously reported for the gum of a tree growing in India, lacking units of 4-O-methylglucuronic acid. Other new side-chain structures were characterized, particularly -alpha-D-Galp-(1-->6)-D-Galp- and alpha-L-Araf-(1-->6)-D-Galp-).

Novel structures in galactoglucomannans of the lichens Cladonia substellata and Cladonia ibitipocae: significance as chemotypes.

The galactoglucomannans of two species of the lichen genus Cladonia, C. substellata and C. ibitipocae, were compared. They were homogeneous on gel-filtration chromatography and structurally related, having (1-->6)-linked alpha-D-mannopyranosyl main-chains, but were substituted in different patterns by alpha- and beta-D-galacto-, beta-D-gluco- and alpha-D-mannopyranosyl groups. The C-1 portions of their 13C-NMR spectra are typical of the lichen species and indicate differences between the two polysaccharides. Partial acetolysis of the galactoglucomannan from C. substellata gave rise to oligosaccharides and three were identified, namely alpha-D-Manp-(1-->3)-alpha beta-D-Galp, alpha-D-Manp-(1-->2)-alpha beta-D-Manp and alpha-D-Manp-(1-->2)-[beta-D-Glcp-(1-->4)]-alpha beta-D-Manp, whereas only the latter two were obtained from that of C ibitipocae. Methylation and Smith degradation data confirmed these results. Whereas the mannobiose represents a common structure in lichen heteropolysaccharides, it is the first time that the other oligosaccharides have been isolated from those of lichens.

Carbohydrate, glycopeptide and protein components of the lichen Sticta sp. and effect of storage.

Three aldoses, six alditols, eight oligosaccharides, three polysaccharides and a glycopeptide were found in a Sticta sp. The most interesting of these were gentiobiose, -triose and -tetraose, O-alpha-D-Galp-(1-->6)-O-beta-D-Galp-(1-->1)-D-glyceritol released on alkaline treatment, and a polysaccharide with predominant (1-->4)-linked beta-D-Xylp units. Present were a galactoglucomannan, a polysaccharide, possibly a mixture, containing beta-D-Galf, and alpha-L-Araf units and a glycopeptide with alpha- and beta-D-Galf, Galp, Glcp and Manp units, having a core with successive (1-->2)-linked alpha-D-Manp residues substituted at O-3 with those of alpha-D-Galf. After four years storage of the lichen, the contents of the principal polyols, ribitol (0.86%), arabinitol (0.99%) and mannitol (2.50%) fell to practically zero, with the appearance of threitol (0.03%), xylitol (0.01%) and myo-inositol (0.03%). Also, the protein content fell from 34 to 18%. The most abundant amino acids were valine (10.5% of total) in fresh lichen and threonine (14.6%) in stored lichen.

The free reducing oligosaccharides of angico branco (Anadenanthera colubrina) gum exudate: an aid for structural assignments in the heteropolysaccharide.

A novel method is described for the determination of sequential side-chain structures in the complex, high-arabinose polysaccharide of the gum exudate of angico branco (Anadenanthera colubrina), using as basis the structurally similar reducing oligosaccharides present in small quantities. Of the ten detected, eight were characterized as disaccharides (2, 3, and 9), linear trisaccharides (1 and 4), branched pentasaccharides (5 and 6), and a doubly branched heptasaccharide (8). The oligosaccharides are substituents of the polysaccharide, which has a (1-->3)-linked beta-D-galactopyranosyl main chain, and with two exceptions they had 6-O-substituted galactopyranosyl reducing ends, probably corresponding to its main-chain units. Characterization was effected through their 1D and 2D NMR correlation spectra, which were better resolved and more readily interpretable than those of the polysaccharide. These spectral data were supported by monosaccharide composition and rotation values. Controlled Smith degradations and methylation analyses were carried out when it was necessary. These data were confirmed by field-desorption MS.

Heteropolysaccharides of the lichen Evernia prunastri.

Extraction of Evernia prunastri with hot aqueous alkali solubilized heteropolysaccharide-containing material which was purified via Fehling precipitation. Further workup of this polysaccharide with Cetavlon gave two fractions having related but different structures. Each structure consisted of a 1-->6-linked alpha-D-Man p main chain partially monosubstituted at O-2 with side chains of alpha-D-Gal p and partially disubstituted at O-2 and O-4 with alpha-D-Gal p and beta-D-Gal p, respectively. The fractions differed in that one contained much more uronic acid than the other, with corresponding predominance of side chains containing alpha-D-Glc pA-(1-->3)-D-Glc p, alpha-D-Gal p-(1-->2)-D-Glc p, and beta-D-Gal f units.

The occurrence of glycolipids in the lichen Ramalina celastri.

We report for the first time the detection of glycolipids in a lichen. Three glycolipids were extracted from Ramalina celastri and their carbohydrates were determined. The main component was O-alpha-D-galactopyranosyl-(1-->6)-O-beta-D-galactopyranosyl-(1-->1)-D- glyceritol, esterified with long-chain fatty acids, some of which were unsaturated.

Cytotoxic effects against HeLa cells of polysaccharides from seaweeds.

Polysaccharides extracted from several red and brown seaweeds growing along the South American coasts were evaluated regarding their cytotoxic effects against HeLa cells. Sulfated galactans were isolated and purified from the red algae Bostrychia montagnei (BHW and B4 fractions, 17 and 22% SO3Na, respectively) and Porphyra columbina (PC75 fraction, 15.6% SO3Na). At maximum concentration of 80 microg/mL, these fractions promoted atypical mitoses in HeLa cells, presence of atypical nuclei and blebs. Alginic acid (160.0 microg/mL) isolated from the brown seaweed Laminaria brasiliensis (molar ratio M/G 1.2) and its M and G blocks (40.0 microg/mL; DP = 20), promoted similar morphologic alterations besides the presence of acidophilic material in the cellular cytoplasm and occurrence of multinuclear cells present in the monolayer. The polysaccharide fraction SF isolated from the brown seaweed Sargassum stenophyllum containing mainly fucose and presenting molar ratio of sulfate/sugar 1.9, promoted very accentuated morphologic modifications in HeLa cells at low concentrations. SF (2.5 microg/mL) caused significant alterations in the cellular morphology and reduction of the growth, such effects being dose dependent. At 40.0 microg/mL, accentuated decrease of the number of mitosis illustrations accompanied by an increase in the number of condensed cells, atypical nuclei, number of clusters and blebs. These results demonstrated that among the anionic polysaccharides tested, the sulfated fucans were those which caused greater cytotoxic effects in HeLa cells.

Cytotoxic effect against HeLa cells of polysaccharides from the lichen Ramalina celastri.

The most active polysaccharides which show anti-tumoral activity are (1-->3)-beta-D-glucans, branched or not at O-6. Since these structures are sometimes poorly soluble in aqueous media, alpha-D-glucans and their chemical derivatives, which are more soluble, were also studied. The present object is to observe morphological alterations in HeLa cells caused by two different polysaccharides obtained from the lichen Ramalina celastri, which are (1-->3),(1-->4)-linked alpha-D-glucan and its sulphated derivative. The cells were incubated in Eagle's medium in the absence or presence of each polysaccharide and routinely processed and analysed by light and electron microscopy. Even though the alpha-D-glucan altered the cellular volume, cytoplasmic densities, and mitosis, the resulting monolayer was similar to the control. TEM analysis showed cytoplasmic blebbing and the presence of an amorphous electron-dense material free in the cytoplasm and interior membranes. The enhanced injury caused by the sulphated derivative was apparent, altering cell adhesion and causing cell aggregation. Nuclear modifications such as fragmentation and condensation of chromatin under the nuclear envelope, which showed to be convoluted, suggested the occurrence of cell death by apoptosis.

Exopolysaccharides, proteins and lipids in Pleurotus pulmonarius submerged culture using different carbon sources.

For many years mushrooms have been consumed and appreciated by their nutritional value, and medicinal properties. The traditional mushroom cultivation takes too long and the macrofungi biotechnology has not been explored in its full potential yet. The goal of this work was to observe if different carbon sources could improve the yield and diversify fungi nutrient composition in submerged culture. Pleurotus pulmonarius mycelia and exopolysacharide productions were evaluated using glucose, galactose, xylose and arabinose. The mycelia yield varied depending on the culture medium, and galactose showed to be the best carbon source to produce EPS. Samples that showed the highest protein contents were grown with xylose (19.44%) and arabinose (26.05%). Furthermore, the biomass cultivated with these carbohydrates and with galactose showed five essential amino acids. All cultured biomass showed low lipid contents (∼1%), being composed mainly of unsaturated fatty acids. All EPS fractions showed as main structures glucans and mannogalactans.

Polysaccharides from peach pulp: structure and effects on mouse peritoneal macrophages.

Pulp from peaches contained polygalacturonic acid and arabinogalactan as main polysaccharides, which were isolated and characterised. The polygalacturonic acid (AE-CWI) contained 95% GalA and its (13)C NMR spectrum showed signals at δ 98.9, 78.0, 71.4, 69.1, 68.4, and 175.1 from C-1, C-4, C-5, C-3, C-2, and C-6 respectively, from (1→4)-linked α-GalpA units. Methylation-MS analysis of carboxy-reduced material (AE-CWI-CR) gave 90% of 2,3,6-Me(3)-galactitol acetate. The arabinogalactan (AE-AG) was composed mainly of Ara (41%) and Gal (50%) and was characterised (methylation analysis and (13)C NMR) as a type II-arabinogalactan. It induced peritoneal macrophage activation in mice, ~70% of cells treated with this fraction (1-50 µg/mL) having morphology of activated cells. However, NO production in macrophages treated with AE-AG was not affected. This suggests a new biological activity for peach polysaccharides.