Isolating conifer DNA: a superior polysaccharide elimination method

Genomic DNA was isolated from frozen needles of maturePinus radiata clones using a modified extraction technique incorporating cetyltrimethylammonium bromide (CTAB) for cell lysis. A high sodium chloride concentration (2 M) was used at 2 stages of the extraction procedure to eradicate polysaccharides, yielding pure genomic DNA suitable for restriction enzyme digestion and PCR amplification. Extractions were scaled down to suit 1.5-mL Eppendorf tubes, allowing easier handling and enhanced sterility.

Antidiabetic properties of polysaccharide- and polyphenolic-enriched fractions from the brown seaweed Ascophyllum nodosum

We screened seaweed species from Atlantic Canada for antidiabetic activity by testing extracts for α-glucosidase inhibitory effect and glucose uptake stimulatory activity. An aqueous ethanolic extract of Ascophyllum nodosum was found to be active in both assays, inhibiting rat intestinal α-glucosidase (IC50 = 77 μg/mL) and stimulating basal glucose uptake into 3T3-L1 adipocytes during a 20-minute incubation by about 3-fold (at 400 μg/mL extract). Bioassay-guided fractionation of the A. nodosum extract showed that α-glucosidase inhibition was associated with polyphenolic components in the extract. These polyphenolics, along with other constituents appeared to be responsible for the stimulatory activity on glucose uptake. However, attempts to further concentrate this activity through fractionation techniques were unsuccessful. A crude polyphenol extract (PPE), an enriched polyphenolic fraction (PPE-F1) and a polysaccharide extract (PSE) were prepared from commercial A. nodosum powder and administered to streptozotocin-diabetic mice for up to 4-weeks by daily gavage at 200 mg/kg body mass. PPE and PPE-F1 improved fasting serum glucose level in diabetic mice; however, the effect was only statistically significant at day 14. In addition, PPE-F1 was shown to blunt the rise in blood glucose after an oral sucrose tolerance test in diabetic mice. Mice treated with PPE and PPE-F1 had decreased blood total cholesterol and glycated serum protein levels compared with untreated diabetic mice, whereas PPE also normalized the reduction in liver glycogen level that occurred in diabetic animals. All 3 A. nodosum preparations improved blood antioxidant capacity.

On a établit une recherche d’un produit anti-diabétique, parmi les algues locales de la région Atlantique du Canada, en examinant la capacité d’un effet inhibiteur de l’enzyme α-glucosidase et une stimulation de l’incorporation cellulaire du glucose. Un extrait éthanol-aqueux de Ascophyllum nodosum nous a donné une activité positive chez les deux essais, une inhibition de l’α-glucosidase provenant de l’intestin du rat (IC50 = 77 μg/mL) et puis une stimulation triple, à une concentration de 400 μg/mL, de l’incorporation du glucose dans les adipocytes 3T3-L1 durant une période de 20 minutes. L’extrait de A. nodosum a été divisé, guidé par les résultats biologiques, et a ainsi démontré la présence d’éléments polyphénoliques associé à l’inhibition de l’α-glucosidase. Ces éléments polyphénoliques ainsi que d’autres semblent être responsables de l’incorporation stimulée du glucose. Il a été impossible de raffiner cette activité lors d’une division des composants. Un extrait brut polyphénolique (PPE), un extrait enrichi polyphénolique (PPE-F1) et puis un extrait polysaccharide (PSE) furent préparés d’une poudre commerciale de A. nodosum et utilisés dans une étude utilisant des souris, rendues diabétiques par injections de streptozotocin, traitées par un gavage journalier de l’extrait 200 mg/kg du poids corporel durant une période de 4 semaines. Le taux du glucose sanguin à jeun des souris fut moins élevé en présence des extraits qu’en leurs absence. Cependant, l’effet était seulement significatif au jour 14. Les résultats étant toutefois variables. En plus, lors d’un essai oral de la tolérance au sucrose chez la souris diabétique, l’extrait PPE-F1 a empêché l’augmentation du taux du glucose sanguin. Les extraits PPE et PPE-F1 ont réduit le taux de cholestérol sanguin et les niveaux de glycation des protéines en comparaison de ces niveaux en absence des extraits tandis que l’extrait PPE a présenté une réduction du niveau de glycogène du foie chez les souris diabétiques. Les trois extraits de A. nodosum ont tous amélioré la capacité antioxidante du sang.

Dietary resistant starch interacts with non-starch polysaccharide and protein to influence colonic protein fermentation, with possible implications for colon cancer risk in humans

Resistant starch is dietary starch that passes undigested into the colon where it can have effects similar to non-starch polysaccharide (dietary fibre). The results support the potential health benefits of incorporating foods containing high levels of both resistant starch and non-starch polysaccharide, such as whole grain breads and legumes, as part of well balanced diets containing fruit, vegetables, cereals, meat/alternatives and dairy foods.

Long-term impact of pneumococcal polysaccharide vaccination on nasopharyngeal carriage in children previously vaccinated with various pneumococcal conjugate vaccine regimes

Previously, the Fiji Pneumococcal Project (FiPP) evaluated reduced dose immunization schedules that incorporated pneumococcal protein conjugate and/or polysaccharide vaccine (PCV7 and 23vPPV, respectively). Immune hyporesponsiveness was observed in children vaccinated with 23vPPV at 12 months of age compared with children who did not receive 23vPPV.

Here we assess the long-term impact of 23vPPV vaccination on nasopharyngeal carriage rates and densities of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus and Moraxella catarrhalis. Nasopharyngeal swabs (n = 194) were obtained from healthy children who participated in FiPP (now aged 5–7 years). S. pneumoniae were isolated and identified by standard culture-based methods, and serotyped using latex agglutination and the Quellung reaction. Carriage rates and densities of S. pneumoniae, H. influenzae, S. aureus and M. catarrhalis were determined using real-time quantitative PCR.

There were no differences in the rate or density of S. pneumoniae, H. influenzae or M. catarrhalis carriage by PCV7 dose or 23vPPV vaccination in the vaccinated participants overall. However, differences were observed between the two main ethnic groups: Fijian children of Indian descent (Indo-Fijian) were less likely to carry S. pneumoniae, H. influenzae and M. catarrhalis, and there was evidence of a higher carriage rate of S. aureus compared with indigenous Fijian (iTaukei) children. Polysaccharide vaccination appeared to have effects that varied between ethnic groups, with 23vPPV vaccination associated with a higher carriage rate of S. aureus in iTaukei children, while there was a lower carriage rate of S. pneumoniae associated with 23vPPV vaccination in Indo-Fijian children.

Overall, polysaccharide vaccination had no long-term impact on pneumococcal carriage, but may have impacted on S. aureus carriage and have varying effects in ethnic groups, suggesting current WHO vaccine schedule recommendations against the use of 23vPPV in children under two years of age are appropriate.

Dietary assimilation and the digestive strategy of the omnivorous anomuran land crab Birgus latro (Coenobitidae).

On Christmas Island, Indian Ocean, the diet of robber crabs, Birgus latro (Linnaeus) was generally high in fat, storage polysaccharides or protein and largely comprised fruits, seeds, nuts and animal material. The plant items also contained significant amounts of hemicellulose and cellulose. In laboratory feeding trials, crabs had similar intakes of dry matter when fed artificial diets high in either fat or storage polysaccharide, but intake was lower on a high protein diet. Assimilation coefficients of dry matter (69–74%), carbon (72–81%), nitrogen (76–100%), lipid (71–96%) and storage polysaccharide (89–99%) were high on all three diets. B. latro also assimilated significant amounts of the chitin ingested in the high protein diet ( 93%) and hemicellulose (49.6–65%) and cellulose (16–53%) from the high carbohydrate and high fat diets. This is consistent with the presence of chitinase, hemicellulase and cellulase enzymes in the digestive tract of B. latro. The mean retention time (27.2 h) for a dietary particle marker (⁵⁷Co-labelled microspheres) was longer than measured in leaf-eating land crabs. The feeding strategy of B. latro involves the selection of highly digestible and nutrient-rich plant and animal material and retention of the digesta for a period long enough to allow extensive exploitation of storage carbohydrates, lipids, protein and significant amounts of structural carbohydrates (hemicellulose, cellulose and chitin).

Monitoring of immune responses to a herbal immuno-modulator in patients with advanced colorectal cancer.

Many herbal medicines are widely used as immuno-modulators in Asian countries. Ganoderma lucidum (Lingzhi) is one of the most commonly used herbs in Asia and preclinical studies have established that the polysaccharide fractions of G. lucidum have potent immuno-modulating effects. However, clinical evidence for this is scanty. The present open-labeled study aimed to evaluate the effects of G. lucidum polysaccharides on selected immune functions in patients with advanced colorectal cancer. Forty-seven patients were enrolled and treated with oral G. lucidum at 5.4 g/day for 12 weeks. Selected immune parameters were monitored using various immunological methods throughout the study. In 41 assessable cancer patients, treatment with G. lucidum tended to increase mitogenic reactivity to phytohemagglutinin, counts of CD3, CD4, CD8 and CD56 lymphocytes, plasma concentrations of interleukin (IL)-2, IL-6 and interferon (IFN)-γ, and NK activity, whereas plasma concentrations of IL-1 and tumor necrosis factor (TNF)-α were decreased. For all of these parameters, no statistical significance was observed when a comparison was conducted between baseline and those values after a 12-week treatment with G. lucidum. The changes of IL-1 were correlated with those for IL-6, IFN-γ, CD3, CD4, CD8 and NK activity (p < 0.05) and IL-2 changes were correlated with those for IL-6, CD8 and NK activity. The results indicate that G. lucidum may have potential immuno-modulating effect in patients with advanced colorectal cancer. Further studies are needed to explore the benefits and safety of G. lucidum in cancer patients.

Antitumor activity and underlying mechamisms of ganopoly, the refined polysaccharides extracted from ganodrema lucidum, in mice

Ganopoly is an aqueous polysaccharide fraction extracted from G. lucidum by patented biochemical technique and has been marketed as an over-the-counter product for chronic diseases including cancer and hepatopathy in many Asian countries. This study was undertaken to explore the anti-tumour effect and the underlying mechanisms of Ganopoly in mice and human tumor cell lines. The maximum tolerateddose (MTD) of Ganopoly in mice was estimated to be 100 mg/kg from a pilot study. Treatment of mice with oral Ganopoly for 10 days significantly reduced the tumour weight of sarcoma-180 in a dose-dependent manner, with inhibition rates of 32.3, 48.2 and 84.9% and growth delays of 1.5, 3.5, and 13.1 days at 20, 50, and 100 mg/kg, respectively. Incubation of Ganopoly at 0.05-1.0 mg/ml for 48 hours showed little or negligible cytotoxicity against human tumor CaSki, SiHa, Hep3B, HepG2, HCT116, HT29, and MCF7 cells in vitro. In contrast, 10 mg/ml of Ganopoly caused significant cytotoxicity in all tumour cells tested except MCF7, with marked apoptotic effects observed in CaSki, HepG2, and HCT116 cells, as indicated by nuclear staining and DNA fragmentation. In addition, Ganopoly enhanced concanavalin A-stimulated proliferation of murine splenocytes by 35.3% at 10 mg/ml, and stimulated the production of nitric oxide in thioglycollate-primed murine peritoneal macrophages in a concentration-dependent manner over 0.05-10 mg/ml. Addition of Ganopoly at 1 mg/ml to murine peritoneal macrophages also potentiated lipopolysaccharide-induced nitric oxide production by 64.2%. Treatment of healthy mice or mice bearing sarsoma-180 with oral Ganopoly over 20-100 mg/kg for 7 day significantly increased the expression of both TNF-α and IFN-γ (at both mRNA and protein levels) in splenocytes in a dose-dependent manner. Moreover, treatment of Ganopoly over 20-100 mg/kg significantly increased cytotoxic T lymphocyte cytotoxicity and NK activity in mice. The overall findings indicated that Ganopoly had antitumor activity with a broad spectrum of immuno-modulating activities and may represent a novel promising immunotherapeutic agent in cancer treatment.

Extruded products with Fenugreek (Trigonella foenum-graecium) chickpea and rice: Physical properties, sensory acceptability and glycaemic index

The present study investigated the effects of fenugreek flour (Trigonella foenum-graecum) and debittered fenugreek polysaccharide (FenuLife^®) inclusion on the physical and sensory quality characteristics, and glycaemic index (GI) of chickpea–rice based extruded products. Based on preliminary evaluation with different proportions of chick pea and rice, a blend of 70:30 chickpea and rice was chosen as the control for further studies. The control blend, replaced with fenugreek flour at 2%, 5% and 10%, or fenugreek polysaccharide at 5%, 10%, 15% and 20%, was extruded at the optimum processing conditions as specified in the detailed study. The extruded products were evaluated for their physical (moisture retention, expansion, hardness, water solubility index (WSI) and water absorption index (WAI)), sensory (flavor, texture, color and overall acceptability) characteristics and in vitro GI to evaluate their suitability as extruded snack products.

Due to the distinct bitter taste, inclusion of fenugreek flour was not acceptable at levels more than 2% in extruded chickpea based products. Addition of fenugreek polysaccharide resulted in slight reduction in radial expansion (P < 0.05), while longitudinal expansion increased. WAI increased while WSI decreased compared to the control (P < 0.05). The mean scores of sensory evaluation indicated that all products containing fenugreek polysaccharide up to 15% were within the acceptable range. There were no significant differences (P > 0.05) between products containing 5–15% fenugreek polysaccharide in their color, flavor, texture and overall quality.

Fenugreek, in the form of debittered polysaccharide (FenuLife^®) could be incorporated up to a level of 15% in a chickpea–rice blend to develop snack products of acceptable physical and sensory properties with low GI Index.

Immunostimulatory polysaccharides from Chlorella pyrenoidosa. A new galactofuranan. Measurement of molecular weight and molecular weight dispersion by DOSY NMR.

Fractionation of the hot water extract of Chlorella pyrenoidosa was performed using a combination of ethanol precipitation, size exclusion chromatography, and anion exchange chromatography. One fraction contained a new polysaccharide, and this compound was shown to be a 1→2-linked β-d-galactofuranan from its 1D and 2D 1H and 13C NMR spectra, with a molecular weight of 15 kDa from DOSY NMR measurements. A number of other fractions were shown to have the same repeating unit as the previously identified arabinogalactan. However, arabinogalactans from different fractions were shown by DOSY NMR to have different molecular weights, which ranged from 27 to 1020 kDa. Agreement with molecular weights measured for some of these fractions by SEC-MALS was very good, further confirming the relationship established by Viel et al. between molecular weights of neutral polysaccharides and self-diffusion coefficients. The smaller molecular weight polysaccharides, the galactofuranan and the 27 and 50 kDa arabinogalactans, were shown to be close to monodisperse by analysis of the distributions of the self-diffusion coefficients for the polymers. The larger arabinogalactans had considerable variation in their molecular weights (188 ± 109 kDa and 1020 ± 370 kDa). Only the two larger arabinogalactans showed immunostimulatory activity.

Immunostimulatory principles from Chlorella pyrenoidosa – part 1 : isolation and biological assessment in vitro

Our proprietary preparation obtained by extraction of Chlorella pyrenoidosa cells, ONC-107 (Respondin™), was recently found to selectively boost antibody response to the influenza vaccine in a human clinical trial. Respondin™ is a potent stimulator of mouse B cell proliferation and an activator of macrophages. Bioactivity-guided resolution concluded that Respondin™ is composed of a mixture of immunostimulatory principles of different chemical nature. A combination of size exclusion, anion exchange and hydrophobic interaction chromatography revealed that the bulk of the immunostimulatory activity resides in polysaccharide/protein complexes with molecular masses larger than 100 kDa that are composed primarily of galactose, rhamnose and arabinose.

Oat beta-glucan lowers total and LDL-cholesterol

Several soluble polysaccharides have been shown to have cholesterol-lowering properties and to have a role in prevention of heart disease. Major sources of one such polysaccharide (beta-glucan) are oats and barley. The aim of this study was to examine the effects on plasma lipid concentrations when beta-glucan derived from a fractionated oat preparation was consumed by people with elevated plasma lipids. A single-blind, crossover design compared plasma cholesterol, triglycerides, high density lipoproteins and low density lipoproteins (LDLs) in 14 people; in the order of low, high and low beta-glucan supplemented diets, each of three weeks duration. For the high beta-glucan diet, an average intake of 7 g per day was consumed from cereal, muffins and bread. The background diet remained relatively constant over the three test periods. Differences during the interventions were calculated by one-way repeated measures analysis of variance. Where treatments were found to be significantly different, pairwise multiple comparison procedures (Tukey Test) were carried out between the high beta-glucan and each of the low beta-glucan phases and there was a highly significant difference between treatments for plasma cholesterol (P = 0.009) and for LDL-cholesterol concentrations (P < 0.001). The differences in plasma cholesterol (6.42 +/- 0.7, 6.14 +/- 0.53, 6.44 +/- 0.67 mmol/L) and LDL-cholesterol (4.59 +/- 0.59, 4.17 +/0.58, 4.52 +/- 0.65 mmol/L) between high beta-glucan and each of the low beta-glucan treatments were significant (P < 0.05). The effect on LDLs (9% lower) is among the highest reported. The results of this study confirm that beneficial reductions in plasma cholesterol and LDL-cholesterol concentrations can be obtained with beta-glucan incorporated into a variety of foods.

Physical, chemical and biological characterization of membrane fouling

In this chapter, advanced characterization of membrane fouling as a diagnostic tool has been summarized to prevent membrane fouling. Physical, chemical and biological analyses as membrane autopsies are mainly utilized to better understand membrane foulant. The physical characterization gives structure, roughness, charge effect, strength and hydrophobicity of membrane fouling. The chemical methods provide qualitative and quantitative measurements of different inorganic and organic matter. The biological properties present the spatial biofilm distribution, structure of dominant microorganisms and isolation and identification of microorganisms. In addition, detailed membrane foulant types are reviewed in terms of structure, roughness, hydrophobicity, charge effect, strength, calcium, magnesium, aluminum, iron, silicate, particle, functional group, biopolymer, humic acid, polysaccharide, structural composition, biofilm structure, microorganism and foulant interaction.

Cellular localization of water soluble, allergenic proteins in rye-grass (Lolium perenne) pollen using monoclonal and specific IgE antibodies with immunogold probes

A postembedding method has been developed for localizing water soluble allergens in rye-grass pollen. This uses dry fixation in glutaraldehyde vapour, followed by 2,2-dimethoxypropane, prior to a 100% ethanol series leading into embedment in LR Gold. This has allowed the attachment of specific monoclonal antibodies to the allergen, which are themselves probed with specific immunogold labels to the antibodies. Wall and cytoplasmic sites have been identified, representing an improvement of fixation and localization of allergens over previous studies employing polyclonal, broad spectrum antibodies.

Rye-grass allergens are labelled in mature pollen grains in the exine (tectum, nexine and central chamber), and in the electron opaque areas of the cytoplasm, especially mitochondria. The allergens are absent from the intine, polysaccharide (P) particles, amyloplasts, Golgi bodies and endoplasmic reticulum. IgE antibodies derived from humans allergic to rye-grass pollen, bind to similar sites in the cytoplasm but only to the outer surface of the pollen grain wall. This method now provides a valuable tool for further developmental studies on the pollen grains, in order to establish the site/s of synthesis of the allergens.

Structural characterisation and bioactivities of hybrid carrageenan-like sulphated galactan from red alga Furcellaria lumbricalis

The red alga, Furcellaria lumbricalis from the coast of the Prince Edward Island (PEI) in Atlantic Canada, was extracted with hot water and fractionated with 0.125. M KCl to obtain a carrageenan-like polysaccharide. The polysaccharide was further purified on ion-exchange and gel-permeation chromatography to yield a fraction (FB1) of uniform size and charge, with an average molecular weight of 428. kD. Oligosaccharides generated with acid hydrolysis of FB1 were sequenced using the electrospray ionisation collision induced dissociation tandem mass spectrometry (ES-CID-MS/MS) technique. On the basis of chemical and spectroscopic analysis, FB1 was characterised to be composed of 1,4-linked 3,6-anhydro-galactose (40%), 1,3-linked 4-sulphated-galactose (30%), 1,3-linked galactose (20%), 1,4-linked galactose (8%) and 1,4-linked 3-O-methyl-galactose (2%), which makes it be a novel sulphated galactan hybrid. The β-secretase (BACE) inhibition and immunomodulation activities of FB1-derived oligosaccharides were evaluated in vitro.

Coassembled nanostructured bioscaffold reduces the expression of proinflammatory cytokines to induce apoptosis in epithelial cancer cells

The local inflammatory environment of the cell promotes the growth of epithelial cancers. Therefore, controlling inflammation locally using a material in a sustained, non-steroidal fashion can effectively kill malignant cells without significant damage to surrounding healthy cells. A promising class of materials for such applications are the nanostructured scaffolds formed by epitope containing minimalist self-assembled peptides (SAPs), as they are bioactive on a cellular length scale, whilst presenting as an easily handled hydrogel. Here, we show that the assembly process distributes an anti-inflammatory polysaccharide, fuccoidan, localised to the nanofibers to function as an anti-inflammatory biomaterial for cancer therapy. We show that it supports healthy cells, whilst inducing apoptosis in cancerous endothelial cells, as demonstrated by the downregulation of the proinflammatory gene and protein expression pathways associated with epithelial cancer progression. Our findings highlight an innovative material approach with potential applications as local epithelial cancer immunotherapy and drug delivery vehicles.