An acidic exopolysaccharide α-D-galacturono-ß-D-glucan produced by the cyanobacterium Scytonema sp.

Some cyanobacteria produce a wide range of secondary metabolites, some of which are of industrial interest. Exopolysaccharides, particularly interesting among them, represent relatively complex primary structures with interesting bioactivity, biodegradability and specific applications. Cultivation of the freshwater cyanobacterium Scytonema sp. provided a proteoglycan-type exopolysaccharide with a relatively low yield and a wide spectrum of molecular weights (Mw) ranging from 2.2 to 1313 × 103 g/mol. Chemical analyses detected the presence of carbohydrates (46 wt%), proteins (10 wt%) and uronic acids (8 wt%). Monosaccharide analysis revealed up to seven neutral sugars with a dominance of glucose (23.6 wt%), galactose (7.4 wt%) and fucose (5.0 wt%) residues, while the others had a much lower content (0.9-3.4 wt%). The presence of galacturonic acid (8.0 wt%) indicated the appearance of ionic type of exopolysaccharide. A preliminary structural study indicated that the α-D-galacturono-ß-D-glucan forms a dominant part of Scytonema sp. exopolymer. Its backbone is composed of two 1,6-linked and one 1,2-linked ß-D-Glcp residues, which is branched at O6 by side chains composed of α-D-GalAp(1 â†’ 2)-ß-D-Glcp(1→ dimer or monomeric ß-D-Glcp(1→ residue.

Dendrobium officinale polysaccharides-chemical properties and pharmacodynamic effects on the airways in experimental conditions.

The study aimed to analyze the effects of Dendrobium polysaccharides on the cough and airway reactivity and compare them with the effects of clinically used antitussives (codeine phosphate and butamirate citrate) and bronchodilators (salbutamol), using the guinea pig test system. Dendrobium officinale polysaccharides contained proteins (4.0 wt%) and phenolic compounds (1.7 wt%) with a molecular weight of 25,000 g/mol. The sugar analysis revealed a dominance of glucose (93.7 wt%) and a lesser amount of mannose (5.1 wt%) while other sugar quantities were negligible. Methylation analysis indicated the presence of highly branched polysaccharides. Glucose was found mainly as terminal, 1,4- and 1,6-linked. Furthermore, some 1,4- and 1,6-linked glucose units were found branched at O2, O3, and O6/O4. Mannose was terminal and 1,4-linked. NMR spectra signals indicate the presence of the (1→4)-linked α-d-glucan, (1→4)-linked ß-d-glucan branched at position O6, (1→6)-linked ß-d-glucan branched at position O3 and (1→4)-linked glucomannan. Pharmacological studies showed statistically significant antitussive activity of Dendrobium polysaccharides, exceeding the effect of clinically used antitussives, which may be partially associated with confirmed bronchodilation and the ability of polysaccharides to increase the threshold of cough receptor activation. Dendrobium polysaccharides may increase the possibility of symptomatic treatment of cough, especially in asthmatics.

Structural properties of the biologically active Dictyosphaerium chlorelloides exopolysaccharide α-d-manno-α-l-rhamno-α-d-(2-O-methyl)-galactan.

Structure of biopolymers produced by microalgae plays an important role for their potential biological activity prediction and applications. Previously isolated and well characterized dominant fractions (Dch5-8) from ion-exchange chromatography separation of the biologically active microalga Dictyosphaerium chlorelloides exopolysaccharide (Dch) were pooled and partially acid hydrolyzed. The dominant sugar components in the combined Dch5-8 fraction were Gal and its 2-O-methyl derivative, Rha and Man, all accounting for about 94 mol% of total amount of sugars. Separation of obtained hydrolysate on Bio-Gel P-2 afforded ten fractions. Their main components were identified by NMR. Based on oligosaccharide structures, the repeating unit of the polysaccharide backbone was identified as →2)-α-L-Rhap-(1→4)-2-O-methyl-[3-O-ß-D-Galp]-α-D-Galp-(1→ branched by Man. Furthermore, the higher molecular weight fraction contained glucuronorhamnan. NMR data indicate 1,4-linked Rha units in the backbone in α and ß configuration, branched at O2 by 2,4-di-O-methyl-ß-d-glucuronic acid.

Investigating the initial steps of auricin biosynthesis using synthetic biology.

Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239) contains a type II polyketide synthase (PKS) biosynthetic gene cluster (BGC) aur1 whose genes were highly similar to angucycline BGCs. However, its product auricin is structurally different from all known angucyclines. It contains a spiroketal pyranonaphthoquinone aglycone similar to griseusins and is modified with D-forosamine. Here, we describe the characterization of the initial steps in auricin biosynthesis using a synthetic-biology-based approach. We have created a plasmid system based on the strong kasOp* promoter, RBS and phage PhiBT1-based integration vector, where each gene in the artificial operon can be easily replaced by another gene using unique restriction sites surrounding each gene in the operon. The system was validated with the initial landomycin biosynthetic genes lanABCFDLE, leading to the production of rabelomycin after its integration into Streptomyces coelicolor M1146. However, the aur1DEFCGHA homologous genes from the auricin aur1 BGC failed to produce rabelomycin in this system. The cause of this failure was inactive aur1DE genes encoding ketosynthases α and ß (KSα, KSß). Their replacement with homologous aur2AB genes from the adjacent aur2 BGC resulted in rabelomycin production that was even higher after the insertion of two genes from the aur1 BGC, aur1L encoding 4-phosphopantetheinyl transferase (PPTase) and aur1M encoding malonyl-CoA:ACP transacylase (MCAT), suggesting that Aur1L PPTase is essential for the activation of the acyl carrier protein Aur1F. These results suggest an interesting communication of two BGCs, aur1 and aur2, in the biosynthesis of the initial structure of auricin aglycone.

The Application of HPLC-FLD and NMR in the Monitoring of Therapy Efficacy in Alpha-Mannosidosis.

BACKGROUND: Alpha-mannosidosis is a rare lysosomal storage disorder, caused by decreased activity of α-D-mannosidase. This enzyme is involved in the hydrolysis of mannosidic linkages in N-linked oligosaccharides. Due to the mannosidase defect, undigested mannose-rich oligosaccharides (Man2GlcNAc - Man9GlcNAc) accumulating in cells are excreted in large quantities in urine. METHODS: In this work, we determined the levels of urinary mannose-rich oligosaccharides in a patient subjected to novel enzyme replacement therapy. Urinary oligosaccharides were extracted using solid phase extraction (SPE), labeled by fluorescent tag 2-aminobenzamide, and quantified by high-performance liquid chromatography (HPLC) with fluorescence detector (FLD). The identity of peaks was determined by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. In addition, the levels of urinary mannose-rich oligosaccharides were also quantified by 1H nuclear magnetic resonance (NMR) spectroscopy. The data were analyzed using one-tailed paired t-test and Pearson's correlation tests. RESULTS: Compared to levels before the administration of therapy, an approximately two-folds decrease in total mannose-rich oligosaccharides after one month of treatment was observed by NMR and HPLC. After four months, an approximately ten-folds significant decrease in total urinary mannose-rich oligosaccharides was detected, suggesting therapy effectiveness. A significant decrease in the levels of oligosaccharides with 7-9 mannose units was detected by HPLC. CONCLUSIONS: The application of both HPLC-FLD and NMR in quantification of oligosaccharide biomarkers is a suitable approach for monitoring of therapy efficacy in alpha-mannosidosis patients.

Structural features of biologically active extracellular polysaccharide produced by green microalgae Dictyosphaerium chlorelloides.

Ion-exchange chromatography of the biologically active extracellular biopolymer produced by D. chlorelloides yielded ten fractions differing in yield, protein content, monosaccharide composition and molecular weight distribution. Their sugar compositional analyses showed rhamnogalactans, substituted to different extent by mannose and glucose, as a dominant EPS component in all fractions (91 %) except one containing arabinogalactan (7 %). In highly branched rhamnogalactans the quantity of linear (1,3-; 1,4- and 1,6-linked) and branched ß-D-galactose units (1,3,6-, 1,4,6- and 1,3,4,6-linked) was nearly equal. From various α-L-rhamnose linkages the 1,2,4-linkage was dominant. Data indicate a rhamnogalactan backbone of EPS, branched by terminal mannose and glucose units, and a lot of O-methylated derivatives of galactose residues (2-O-methyl, 2,3-O-dimethyl, 3-O-methyl and 6-O-methyl). In individual fractions their content and type varied. Detail study of the arabinogalactan showed that its backbone consists of 1,3-linked ß-D-Galp units; some of them are branched through O-4 by 6-OMe-α-D-Galp- (1 â†’ 2) -α-L-Araf side chain, other through O-6 by 3-OMe-ß-D-Galp, 6-OMe-ß-D-Galp, ß-D-Galp and ß-D-Galf.

Lactylated acidic exopolysaccharide produced by the cyanobacterium Nostoc cf. linckia.

Cyanobacteria produce a wide range of metabolites of interest for industrial or medical use. The cultivation of freshwater Nostoc cf. linckia yielded 5.4 g/L of a crude exopolysaccharide (cEPS) with a molecular weight of 1.31 × 105 g/mol. Ion-exchange chromatography of cEPS yielded two dominant fractions, EPS-1 and EPS-2, differing in molecular weight. The lower molecular weight fraction (EPS-1) was subjected to structural studies. Results of chemical and spectroscopic analyses showed that three of the four dominant sugars, glucose, galactose and xylose are 1,4-linked in the backbone in the following order: [→4)-ß-D-Xylp-(1 â†’ 4)-ß-D-Glcp-(1 â†’ 4)-α-D-Galp-(1 â†’ 4)-ß-D-Glcp-(1→]n. Terminal mannose residues were identified as side chains linked at C3 of every third backbone xylose and every second glucose is branched at C6 by 3-O-lactyl-ß-D-glucuronic acid (nosturonic acid). Antioxidant properties of EPS were tested using two in vitro methods. Both assays showed that the cEPS was more active than purified EPS-1 and EPS-2 fractions and deproteinized EPS.

Optimizing acid hydrolysis for monosaccharide compositional analysis of Nostoc cf. linckia acidic exopolysaccharide.

The exact estimation of monosaccharide composition is important in the primary structure elucidation of polysaccharides. An acid hydrolysis is usually performed for glycosidic bonds cleavage and releasing of monosaccharides. In this study, optimal conditions of total acid hydrolysis using trifluoroacetic acid (TFA) of acidic lactylated Nostoc cf. linckia exopolysaccharide (EPS) were investigated by NMR spectroscopy. Results of a series of experiments with modified acid concentration, temperature and time of hydrolysis, have shown 2 M TFA, 110 °C, 3 h as the most optimal. The stability of EPS monosaccharide components was also explored. Low stability was found at all tested conditions already during the first hour of hydrolysis; all neutral monosaccharides were degraded from 25% to 40% and glucuronic acid to 75%. NMR, contrary to standard techniques used in monosaccharide compositional analysis (HPLC, HPAEC), allowed simultaneous quantification of all GlcA forms; the free one, that one linked in oligosaccharides, as well as GlcA degradation product γ-lactone. NMR as detection method improves information about uronic acid content in EPS.

An efficient system for stable markerless integration of large biosynthetic gene clusters into Streptomyces chromosomes.

The bacteria of the genus Streptomyces are among the most important producers of biologically active secondary metabolites. Moreover, recent genomic sequence data have shown their enormous genetic potential for new natural products, although many new biosynthetic gene clusters (BGCs) are silent. Therefore, efficient and stable genome modification techniques are needed to activate their production or to manipulate their biosynthesis towards increased production or improved properties. We have recently developed an efficient markerless genome modification system for streptomycetes based on positive blue/white selection of double crossovers using the bpsA gene from indigoidine biosynthesis, which has been successfully applied for markerless deletions of genes and BGCs. In the present study, we optimized this system for markerless insertion of large BGCs. In a pilot test experiment, we successfully inserted a part of the landomycin BGC (lanFABCDL) under the control of the ermEp* promoter in place of the actinorhodin BGC (act) of Streptomyces lividans TK24 and RedStrep 1.3. The resulting strains correctly produced UWM6 and rabelomycin in twice the yield compared to S. lividans strains with the same construct inserted using the PhiBT1 phage-based integration vector system. Moreover, the system was more stable. Subsequently, using the same strategy, we effectively inserted the entire BGC for mithramycin (MTM) in place of the calcium-dependent antibiotic BGC (cda) of S. lividans RedStrep 1.3 without antibiotic-resistant markers. The resulting strain produced similar levels of MTM when compared to the previously described S. lividans RedStrep 1.3 strain with the VWB phage-based integration plasmid pMTMF. The system was also more stable. KEY POINTS: • Optimized genome editing system for markerless insertion of BGCs into Streptomyces genomes • Efficient heterologous production of MTM in the stable engineered S. lividans strain.

Structural features of the bioactive cyanobacterium Nostoc sp. exopolysaccharide.

Microalgal biopolymers are studied mainly in terms of physico-chemical characterization, biological effects as well as possible biotechnological applications. Due to the significant antitussive, bronchodilator, anti-inflammatory and immunomodulatory effects of the previously isolated crude extracellular polysaccharide (EPS) produced by the cyanobacterium Nostoc sp., the purified biopolymer and its oligosaccharides, obtained after partial acid hydrolysis, were subjected to an in-depth NMR structural study. Analyses of the data obtained by chemical methods and NMR showed that the EPS backbone is composed of the repeating unit [→4)-ß-D-Xylp-(1 → 4)-ß-D-Glcp-(1 → 4)-α-L-Arap-(1 → 3)-ß-D-Manp-(1→]n, in which about 60% of glucose units are substituted at C6 by uronic acids, in particular by the unusual unsaturated 3-O-lactyl-4-deoxy-α-erythro-hex-4-enopyranuronic acid, and to a lesser extent by ß-D-glucuronic acid and 3-O-lactyl-ß-D-glucuronic acid. These findings, structural features and identified biological effects, suggest the potential use of this biopolymer in the medical-pharmaceutical field.

Chlorella vulgaris α-L-arabino-α-L-rhamno-α,ß-D-galactan structure and mechanisms of its anti-inflammatory and anti-remodelling effects.

Microalgal exopolysaccharides (EPSs) are given great attention due to their potential biotechnology applications. Purified C. vulgaris EPS was subjected to compositional and sugar linkage analyses, and partial acid hydrolysis. Hydrolysate separation by gel chromatography afforded oligosaccharide fractions. Both, EPS and oligomers were studied by NMR spectroscopy. Data suggest very complex highly branched α-L-arabino-α-L-rhamno-α,ß-D-galactan structure. Backbone repeating unit is formed by →2)-α-L-Rha (1 â†’ 3)-α-L-Rha(1 â†’ sequence, highly branched by long 1,6-linked α-D-Galp side chains, further branched at C2, C3 or C4 by α-L-Araf, α-D-Galf and ß-D-Galf residues. α-L-Araf form longer 1,2-linked chains branched at C3, C4 or C5. Galf residues are localized as terminal units predominantly in the ß configuration, while α-D-Galp and α-L-Araf may be partially O-methylated. Ex vivo biological assays showed increased interleukin-12 (IL-12) and interferon-gamma (INF-γ) levels corresponding to transforming growth factor beta (TGF-ß) decrease in guinea pig model experimental asthma. These facts point to the anti-remodelling effect of Chlorella EPS and suggest its possible application in the treatment of asthma and chronic obstructive pulmonary disorder.

Structural characteristics and biological effects of exopolysaccharide produced by cyanobacterium Nostoc sp.

Complex structure of cyanobacterium Nostoc sp. exopolysaccharide (EPS), with apparent molecular weight 214 × 103 g/mol, can be deduced from its composition. Chemical and NMR analyses found four dominant sugar monomers, namely (1 → 4)-linked α-l-arabinopyranose, ß-d-glucopyranose, ß-d-xylopyranose and (1 → 3)-linked ß-d-mannopyranose, two different uronic acids and a lactyl group, with (1 → 4,6)-linked ß-d-glucopyranose as the only branch point suggest a complex structure of this polymer. The dominant uronic acid is α-linked, but it remained unidentified. ß-d-Glucuronic acid was present in lower amount. Their position as well as that of lactyl remained undetermined too. Different doses of orally administered EPS in guinea pigs evoked a significant decrease in cough effort and a decrease in airway reactivity. The antitussive efficacy and bronchodilator effect of higher doses of EPS were found to be similar to that of the antitussive drug codeine and the antiasthmatic salbutamol. Without significant cytotoxicity on the RAW 264.7 cells, EPS stimulated the macrophage cells to produce pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and prostaglandins (PGs) and nitric oxide (NO) via induction of COX-2 and iNOS expression, respectively, suggesting that this biopolymer potentiates an early innate immune response and can therefore be used as a new immune modulator.

A Structural Analysis of the Angucycline-Like Antibiotic Auricin from Streptomyces lavendulae Subsp. Lavendulae CCM 3239 Revealed Its High Similarity to Griseusins.

We previously identified the aur1 gene cluster in Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239), which is responsible for the production of the angucycline-like antibiotic auricin (1). Preliminary characterization of 1 revealed that it possesses an aminodeoxyhexose d-forosamine and is active against Gram-positive bacteria. Here we determined the structure of 1, finding that it possesses intriguing structural features, which distinguish it from other known angucyclines. In addition to d-forosamine, compound 1 also contains a unique, highly oxygenated aglycone similar to those of spiroketal pyranonaphthoquinones griseusins. Like several other griseusins, 1 also undergoes methanolysis and displays modest cytotoxicity against several human tumor cell lines. Moreover, the central core of the aur1 cluster is highly similar to the partial gris gene cluster responsible for the biosynthesis of griseusin A and B in both the nature of the encoded proteins and the gene organization.

The polyphenolic-polysaccharide complex of Agrimonia eupatoria L. as an indirect thrombin inhibitor - isolation and chemical characterization.

The polyphenolic-polysaccharide complex was isolated from the dried aerial parts of the medicinal plant Agrimonia eupatoria L. using a multi-step process involving the degreasing of the plant material by extraction with organic solvents, followed by extraction with hot alkali, neutralization, further separation with organic solvents and dialysis. The complex was homogeneous with a molecular weight of about 55 × 103 g/mol and consisted mainly of carbohydrates and polyphenols matrix, composed of lignin-related units, with the dominance of dimethoxyphenyl structures. The carbohydrate moiety consists mostly of arabinogalactan associated with highly esterified rhamnogalacturonan. In vitro anticoagulant studies revealed the ability of the A. eupatoria complex to inhibit plasma clot formation, mainly in the intrinsic pathway of the blood coagulation cascade. Further studies on the mechanisms of this anticoagulant activity revealed that the isolate was primarily an indirect inhibitor of thrombin, mediated by antithrombin or by heparin cofactor II. Such mechanism of action is characteristic for highly sulfated glycosaminoglycans.

Chemico-physical and pharmacodynamic properties of extracellular Dictyosphaerium chlorelloides biopolymer.

Microalgae occupy all territories and their products represent a rich source of phytochemicals for human being. Green microalga Dictyosphaerium chlorelloides was found to be a significant producer of the extracellular biopolymer. Dominant components of the biopolymer were found to be Gal (39 wt%) with its methyl derivatives (15 wt%), Rha (19 wt%) and Man (14 wt%). 2-OMe-Gal was found to be the major derivative while other sugars, namely 3-OMe-, 6-OMe- and 2,3-di-OMe-Gal, 3-OMe-Glc and 4-OMe-Xyl were in smaller amounts. NMR spectroscopy revealed complex structure with galactan backbone branched by sugars in furano and pyrano forms in alpha and beta configurations. NMR data of 2-OMe, 3-OMe, 2,3-OMe and 6-OMe galactoses afforded characteristic values for O-methyls in each position. Biopolymer antitussive effect was similar to that of centrally acting antitussive drugs, indicating its relatively good antitussive potential.

Polyphenolic-polysaccharide conjugates of Sanguisorba officinalis L. with anticoagulant activity mediated mainly by heparin cofactor II.

A macromolecular complex has been isolated from the dried flowering parts of medicinal plant Sanguisorba officinalis L. (So) by multi-step extraction procedure, including that with extraction by organic solvents to degrease the plant material, then with hot alkali, followed by neutralization, partitioning with organic solvents and dialysis. The complex was purified by size-exclusion chromatography into five fractions labeled as So1-So5. Individual fractions differed in the chemical composition and molecular weight distribution patterns. In vitro anticoagulant activity tests showed in all fractions more or less important inhibition of plasma clots, however, So3 and So4 were the most active. The anticoagulant activity of So3 was even more significant than that of the unfractionated complex So. These S. officinalis conjugates were able to inhibit mainly the activity of thrombin when they were mediated by heparin cofactor II, but what was unexpected they were the non-direct inhibitors of factor Xa, mediated by antitrombin, where such mechanism of action is typical for a highly sulphated glycosaminoglycans.

Echinacea complex--chemical view and anti-asthmatic profile.

ETHNOPHARMACOLOGICAL RELEVANCE: Echinacea purpurea (L.) Moench is one of the mostly used herbs in the traditional medicine for the treatment of respiratory diseases. Modern interest in Echinacea is directed to its immunomodulatory activity. Recent studies have shown that secretion of asthma-related cytokines in the bronchial epithelial cells can be reversed by Echinacea preparations. AIM OF THE STUDY: To examine the pharmacodynamics profile of Echinacea active principles, a complex has been isolated from its flowers by alkaline extraction and has been tested using an animal model of allergic asthma. MATERIAL AND METHODS: The structural features of Echinacea purpurea complex was determined using chemical and spectroscopic methods. Allergic inflammation of the airways was induced by repetitive exposure of guinea pigs to ovalbumin. Echinacea complex was then administered 14 days in 50mg/kg b.w. daily dose perorally. Bronchodilatory effect was verified as decrease in the specific airway resistance (sRaw) in vivo and by reduced contraction amplitude (mN) of tracheal and pulmonary smooth muscle to cumulative concentrations of acetylcholine and histamine in vitro. The impact on mucociliary clearance evaluated measurement of ciliary beat frequency (CBF) in vitro using LabVIEW™ Software. Anti-inflammatory effect of Echinacea complex was verified by changes in exhaled NO levels and by Bio-Plex® assay of Th2 cytokine concentrations (IL-4, IL-5, IL-13 and TNF-alpha) in serum and bronchoalveolar lavage fluid (BALF). RESULTS: Chemical and spectroscopic studies confirmed the presence of carbohydrates, phenolic compounds and proteins, as well as the dominance of rhamnogalacturonan and arabinogalactan moieties in Echinacea complex. The significant decrease in sRaw values and suppressed histamine and acetylcholine-induced contractile amplitude of isolated airways smooth muscle that were similar to effects of control drug salbutamol confirmed Echinacea complex bronchodilatory activity. The anti-inflammatory effect was comparable with that of control agent budesonide and was verified as significantly reduced exhaled NO levels and concentration of Th2 cytokines in serum and BALF. The values of CBF were changed only insignificantly on long-term administration of Echinacea complex suggested its minimal negative impact on mucociliary clearance. CONCLUSION: Pharmacodynamic studies have confirmed significant bronchodilatory and anti-inflammatory effects of Echinacea complex that was similar to effects of classic synthetic drugs. Thus, results provide a scientific basis for the application of this herb in traditional medicine as a supplementary treatment of allergic disorders of the airways, such as asthma.

Biotransformation of various saccharides and production of exopolymeric substances by cloud-borne Bacillus sp. 3B6.

The ability of Bacillus sp. 3B6, a bacterial strain isolated from cloudwaters, to biotransform saccharides present in the atmosphere was evaluated using in situ 1D and 2D NMR spectroscopy. Bacillus is one of the genera most frequently described in the air and in atmospheric waters. Sugars present in these environments have a biogenic origin; they include alditols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Bacillus sp. 3B6 was able to efficiently metabolize sugars, which could thus provide sources of energy for this bacterium and allow it to live and to be metabolically active in warm clouds. In addition, a number of these saccharides (L-arabitol, D-fructose, sucrose, D-glucose, cellotetraose, cellulose, and starch) were transformed to EPSs (exopolymeric substances). We have clearly identified the structure of two EPSs as 1,6-α-galactan and partially acetylated polyethylene glycol. 1,6-α-Galactan is a newly described polymer. The production of EPSs might protect this bacterium under hostile cloud environment conditions, including low nutrient availability, cold temperature and freeze-thaw processes, UV and radical exposure, and evaporation-condensation processes and thus desiccation and osmolarity changes. EPSs could also have a potential role in atmospheric processes because they can be considered as secondary organic aerosols and efficient cloud condensation nuclei.

Coffea arabica instant coffee--chemical view and immunomodulating properties.

Results of chemical analyses and immunological studies of two Coffea arabica instant coffee powders obtained by freeze-dried (ICPf) and spray-dried (ICPs) procedures, and arabinogalactan-protein (AGP3) obtained from ICPf are presented. For instant coffee powders no significant differences have been found in carbohydrate (ICPf: 37%, ICPs: 38%) as well as in caffeine (ICPf: 3.0%, ICPs: 3.4%) contents. Their (1)H NMR spectra revealed differences in trigonelline and chlorogenic acids content and in a degree of AGP backbone substitution. Immunobiological tests of all samples (ICPf, ICPs, AGP2 and AGP3) revealed a significant immunostimulatory effect on induction of interleukin 2 and free radicals secretion by mice immunocytes. Moreover, tests revealed more pronounced effect of arabinogalactans AGP2 and AGP3 compared to instant coffee powders (ICPf and ICPs).

Effects of extraction condition on structural features and anticoagulant activity of F. vesca L. conjugates.

From the air-dried Wild strawberry (Fragaria vesca L., family Rosaceae) leaves five water-soluble glycoconjugates Fv I-V by different extraction conditions have been isolated. Effects of extraction steps/agents on chemical composition and anticoagulant activity of Fv I-V were examined. Dark brown F. vesca conjugates Fv I-V were recovered in 4.5-8.4% yields, based on dry herb. Isolates were composed of carbohydrate, phenolic and protein components. Fv I-V displayed on HPLC broad molecule-mass distribution patterns with dominance of low molecule-masses 9-14 kDa. Their carbohydrate parts revealed high hexuronic acids content (35-60%) while the dominant neutral sugars - galactose, arabinose and rhamnose were found in lower amounts and indicated the presence of rhamnogalacturonans associated with arabinogalactans in all F. vesca preparations. In all Fv I-V isolates high polyphenolic contents were determined, whereas proteins were found in low amounts only. In in vitro experiments on human pooled plasma Fv I-V showed at higher concentrations complete inhibition of plasma clot formation and the most active conjugates in aPTT, PT and TT tests were shown to be Fv I and Fv III, containing the highest amounts of phenolics.