Yacon fructans (Smallanthus sonchifolius) extraction, characterization and activation of macrophages to phagocyte yeast cells.

Yacon (Smallanthus sonchifolius) originates from the Andean region and has spread across South America, Europe and Japan. In contrast with most roots, yacon stores its carbohydrates in fructooligosaccharides (FOS) and contains approximately 37% of FOS in its root dry matter. Aqueous extracts of yacon were characterized through TLC, methylation, NMR, and ESI-MS. FOS of yacon showed as linear fructooligosaccharides containing almost exclusively (2→1)-linked ß-fructofuranosyl units, with terminal α-glucopyranosyl and ß-fructofuranosyl units. ESI-MS analyses indicated a wide degree of polymerization (DP) ranging from 2 to 10. The effect of the isolated FOS on non-specific immune activity by THP-1 cells was evaluated through phagocytic activity against heat-killed yeast (Saccharomices cerevisiae). The stimulant effect of yacon FOS was dose- and time-dependent, showing results more effective than branched FOS observed in previous studies. The results reinforce the use of linear yacon FOS as immunomodulators.

Phytochemical analysis and anti-inflammatory evaluation of compounds from an aqueous extract of Croton cajucara Benth.

Croton cajucara Benth. is a medicinal plant popularly used in the Brazilian Amazonia, where it is known as sacaca, being consumed as tea, decoction or infusion of the leaves and stem bark. From a decoction of the leaves, a comprehensive phytochemical analysis was developed by liquid chromatography-mass spectrometry. Many compounds were identified for the first time in C. cajucara, such as O-glycosides of kaempferol and quercetin, flavonoid-C-glycosides, tannins and cinnamic acid derivatives. These compounds were fractionated by polarity and assayed for their anti-inflammatory activity, using a model of mice edema, induced by an intraplantar injection of carrageenan. All fractions exhibited anti-inflammatory properties.

Chemical characterization of heteropolysaccharides from green and black teas (Camellia sinensis) and their anti-ulcer effect.

In order to obtain polysaccharides from green and black teas (Camellia sinensis), commercial leaves were submitted to infusion and then to alkaline extraction. The extracts were fractionated by freeze-thawing process, giving insoluble and soluble fractions. Complex arabinogalactan protein from the soluble fractions of both teas (GTPS and BTPS) were determined by methylation analysis and (1)H/(13)C-HSQC spectroscopy, showing a main chain of (1→3)-ß-Galp, substituted at O-6 by (1→6)-linked ß-Galp with side chains of α-Araf and terminal units of α-Araf, α-Fucp and α-Rhap. A highly branched heteroxylan from the insoluble fractions (GTPI and BTPI) showed in methylation analysis and (1)H/(13)C-HSQC spectroscopy the main chain of (1→4)-ß-Xylp, substituted in O-3 by α-Araf, ß-Galp and α-Glcp units. Evaluating their gastroprotective activity, the fractions containing the soluble heteropolysaccharides from green (GTPS) and black teas (BTPS) reduced the gastric lesions induced by ethanol. Furthermore, the fraction of insoluble heteropolysaccharides of green (GTPI) and black (BTPI) teas also protected the gastric mucosa. In addition, the maintenance of gastric mucus and reduced glutathione (GSH) levels was involved in the polysaccharides gastroprotection.

Baccharis dracunculifolia-based mouthrinse alters the exopolysaccharide structure in cariogenic biofilms.

Baccharis dracunculifolia is a native plant from Brazil with antimicrobial activity. The purpose of this study was to investigate whether a B. dracunculifolia-based mouthrinse (Bd) changes the structure of insoluble exopolysaccharides (IEPS) in Streptococcus mutans UA159 cariogenic biofilm. Biofilms were grown on glass slides and treated with Bd, its vehicle (VC), chlorhexidine digluconate (CHX), or saline solution (NaCl). Among the treatments, only CHX significantly reduced the biofilm biomass and bacterial viability (p<0.05). Gas chromatography-mass spectrometry and nuclear magnetic resonance analyses revealed that IEPS from the four biofilm samples were α- glucans containing different proportions of (1→6) and (1→3) glycosidic linkages. The structural differences among the four IEPS were compared by principal component analysis (PCA). PCA analysis indicated that IEPS from VC- and NaCl-treated biofilms were structurally similar to each other. Compared with the control, IEPS from Bd- and CHX-treated biofilms were structurally different and had distinct chemical profiles. In summary, the fact that Bd changed the IEPS chemical composition indicates that this mouthrinse may affect the cariogenic properties of the S. mutans biofilm formed.

Genetic and chemical diversity in seeds of cactus mandacaru (Cereus sp.) from two edaphoclimatic regions contrasting.

The purpose of this study was to evaluate the chemical, physiological and genetic differences in seeds of cactus of the Cereus genus (mandacaru) cultivated in the Northeast (Picos, State of Piauí) and Southern (Maringá, State of Paraná) regions of Brazil. Over a period of eight days, temperatures of 25°C and 30°C were equally efficient for the germination of all the seeds. Oleic acid (C18:1) was the most common fatty acid found in the seeds collected in the Southern (41%) and Northeast (45.5%) regions. The analysis of lipases indicated that seeds from Maringá have high mean observed and expected heterozygosities and that seeds from Picos have a higher number of alleles per loci. Therefore, the seeds of mandacaru from the semiarid region of Northeast as well as the seeds from the South (the two contrasting regions of Brazil) are promising with regards to the preservation of the biodiversity in the genome of mandacaru. The low genetic identity between mandacaru seeds from Maringá and Picos at Lipase-5 locus analysis (I = 0.77) suggests that the mandacaru plants from Maringá and Picos may correspond to two species: C. peruvianus and C. jamacaru, respectively.

Glycan analysis of Fonsecaea monophora from clinical and environmental origins reveals different structural profile and human antigenic response.

Dematiaceous fungi constitute a large and heterogeneous group, characterized by having a dark pigment, the dihydroxynaftalen melanin-DHN, inside their cell walls. In nature they are found mainly as soil microbiota or decomposing organic matter, and are spread in tropical and subtropical regions. The fungus Fonsecaea monophora causes chromoblastomycosis in humans, and possesses essential mechanisms that may enhance pathogenicity, proliferation and dissemination inside the host. Glycoconjugates confer important properties to these pathogenic microorganisms. In this work, structural characterization of glycan structures present in two different strains of F. monophora MMHC82 and FE5p4, from clinical and environmental origins, respectively, was performed. Each one were grown on Minimal Medium (MM) and Czapeck-Dox (CD) medium, and the water soluble cell wall glycoconjugates and exopolysaccharides (EPS) were evaluated by NMR, methylation and principal component analysis (PCA). By combining the methylation and 2D NMR analyses, it was possible to visualize the glycosidic profiles of the complex carbohydrate mixtures. Significant differences were observed in ß-D-Galf-(1→5) and (1→6) linkages, α- and ß-D-Glcp-(1→3), (1→4), and (1→6) units, as well as in α-D-Manp. PCA from (1)H-NMR data showed that MMHC82 from CD medium showed a higher variation in the cell wall carbohydrates, mainly related to O-2 substituted ß-D-Galf (δ 106.0/5.23 and δ 105.3/5.23) units. In order to investigate the antigenic response of the glycoconjugates, these were screened against serum from chromoblastomycosis patients. The antigen which contained the cell wall of MMHC82 grown in MM had ß-D-Manp units that promoted higher antigenic response. The distribution of these fungal species in nature and the knowledge of how cell wall polysaccharides and glycoconjugates structure vary, may contribute to the better understanding and the elucidation of the pathology caused by this fungus.

Anti-inflammatory properties of the medicinal mushroom Cordyceps militaris might be related to its linear (1→3)-ß-D-glucan.

The Ascomycete Cordyceps militaris, an entomopathogenic fungus, is one of the most important traditional Chinese medicines. Studies related to its pharmacological properties suggest that this mushroom can exert interesting biological activities. Aqueous (CW and HW) and alkaline (K5) extracts containing polysaccharides were prepared from this mushroom, and a ß-D-glucan was purified. This polymer was analysed by GC-MS and NMR spectrometry, showing a linear chain composed of ß-D-Glcp (1→3)-linked. The six main signals in the 13C-NMR spectrum were assigned by comparison to reported data. The aqueous (CW, HW) extracts stimulated the expression of IL-1ß, TNF-α, and COX-2 by THP-1 macrophages, while the alkaline (K5) extract did not show any effect. However, when the extracts were added to the cells in the presence of LPS, K5 showed the highest inhibition of the pro-inflammatory genes expression. This inhibitory effect was also observed for the purified ß-(1→3)-D-glucan, that seems to be the most potent anti-inflammatory compound present in the polysaccharide extracts of C. militaris. In vivo, ß-(1→3)-D-glucan also inhibited significantly the inflammatory phase of formalin-induced nociceptive response, and, in addition, it reduced the migration of total leukocytes but not the neutrophils induced by LPS. In conclusion, this study clearly demonstrates the anti-inflammatory effect of ß-(1→3)-D-glucan.

Structural studies of an exopolysaccharide produced by Gluconacetobacter diazotrophicus Pal5.

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium that has been found colonizing several plants. This acid-tolerant bacterium produces phytohormones that promote plant growth and is also able to grow in high-sugar concentrations. It has been demonstrated that exopolysaccharides (EPS), which are produced by strain Pal5 of G. diazotrophicus, play an important role in plant infection. We have investigated the structure of the EPS, which was produced by a strain of Pal5 grown in liquid medium containing mannitol as the sole carbon source. The results reveal an EPS with Glc, Gal, Man in a molar ratio of 6:3:1, respectively. NMR spectroscopy and chemical derivatization have revealed that the EPS structure has 4-O-substituted units of ß-glucose, 3-O-substituted units of ß-galactose and 2-O-substituted units of α-mannose. Glucose and galactose units linked at C6 were also found. The structure proposed herein is different from EPS produced by other species of Gluconacetobacter published to date.

Galactofuranosyl glycosides: immunomodulatory effects on macrophages and in vivo enhancement of lethality on sepsis.

Galactofuranoside derivatives were synthesised by the classic Fischer glycosydation method, and their immune modulation properties were studied in vitro and in vivo. NMR spectroscopic and ESI-MS analyses confirmed the purity and authenticity of all derivatives. Their phagocyte capacities were tested in resident macrophages. Methyl ß-galactofuranoside (GFB-Me) and n-octyl ß-galactofuranoside (GFB-O) had an immune stimulant effect at 25µmolml(-1) with an enhancement of 35.12%±0.06 SD and 17.49%±0.11 SD, respectively, but Methyl α-galactofuranoside (GFA-Me) and n-octyl α-galactofuranoside (GFA-O) gave a low immune response. Methyl α-galactofuranoside 5,6-O-isopropylidene (GFA-IP) and Methyl ß-galactofuranoside 5,6-O-isopropylidene (GFB-IP) had negative values relative to the control group of minus 4.96%±0.10 SD and -40.72%±0.07 SD, respectively. Furthermore, GFB-Me and GFB-Me-IP were evaluated in vivo on the lethality induced by cecal ligation and puncture. Cytokine levels and iNOS expression were determined and correlated to mortality data. The results showed that the free HO-5 and HO-6 and the ß-configuration are essential for the induction of phagocytic activity by the galactofuranosyl units. The methyl ß-galactofuranosides also enhanced lethality during sepsis, increasing the levels of pro-inflammatory cytokines and iNOS expression.

A robust method to quantify low molecular weight contaminants in heparin: detection of tris(2-n-butoxyethyl) phosphate.

Recently, oversulfated chondroitin sulfate (OSCS) was identified in contaminated heparin preparations, which were linked to several adverse clinical events and deaths. Orthogonal analytical techniques, namely nuclear magnetic resonance (NMR) and capillary electrophoresis (CE), have since been applied by several authors for the evaluation of heparin purity and safety. NMR identification and quantification of residual solvents and non-volatile low molecular contaminants with USP acceptance levels of toxicity was achieved 40-fold faster than the traditional GC-headspace technique, which takes ~120 min against ~3 min to obtain a (1)H NMR spectrum with a signal/noise ratio of at least 1000/1. The procedure allowed detection of Class 1 residual solvents at 2 ppm and quantification was possible above 10 ppm. 2D NMR techniques (edited-HSQC (1)H/(13)C) permitted visualization of otherwise masked EDTA signals at 3.68/59.7 ppm and 3.34/53.5 ppm, which may be overlapping mononuclear heparin signals, or those of ethanol and methanol. Detailed NMR and ESI-MS/MS studies revealed a hitherto unknown contaminant, tris(2-n-butoxyethyl) phosphate (TBEP), which has potential health risks.