Low Molecular Weight Polysaccharides Research Articles

Preparation of low molecular weight Sargassum fusiforme polysaccharide and its anticoagulant activity

Heparin has been used as an anticoagulant drug for many years, but it has significant side effects. In the search for good substitutes, low molecular weight (MW) polysaccharides from Sargassum fusiforme have been examined and confirmed to possess biological activities. Here, S. fusiforme polysaccharides (SFP) were extracted and subjected to a hydrogen peroxide (H2O2) oxidation method for the preparation of low-MW SFP (LSFP). The effects of temperature, pH, and H2O2 concentration on the degradation process were also examined. Several LSFP of 36, 9, 5.7, and 2.7 kDa were obtained under different conditions, and their anticoagulant activities studied in vitro. The results showed that SFP and LSFP prolonged activated partial thromboplastin (APTT), prothrombin (PT) and thrombin times (TT) significantly, indicating that these low MW polysaccharides possessed anticoagulant activity in the intrinsic, extrinsic, and common coagulation pathways. As these effects were related to the MW of the polysaccharides in APTT and TT but not in PT, the contents of the monosaccharide fucose and sulfate and the polysaccharide MW could have exerted combined effects. The details of this mechanism require further verification.

Determination of Polysaccharide Hydrolyzates in Chinese Herbal Medicine by UltraHigh-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry and Evaporative Light Scattering Detection

ABSTRACTUltrahigh-performance liquid chromatography coupled with evaporative light scattering detector was used to optimize acid hydrolysis conditions of polysaccharides from Dendrobium candidum. The optimized conditions were the concentration of H2SO4, 0.4 M, hydrolysis temperature, 90°C, and hydrolysis time, 1 h. Ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used to obtain the total ion current chromatograms and determine the hydrolyzates of polysaccharides, such as monosaccharides, oligosaccharides, and some low-molecular weight polysaccharides from D. candidum, Cordyceps sinensis, and Ganoderma atrum. The accurate mass data were analyzed by chemometric tools, including an unsupervised analytical method, principal component analysis (one-way ANOVA, p < 0.05), and five supervised analytical methods, partial least square discriminant analysis, support vector machine, naïve Bayes, decision tree, and neural network. The results showed that D. candidum polysaccharides from different locations and grades can be discriminated effectively by the principal component analysis scores. The quality predictive models showed a high value with a low prediction error calculated from both validation and training sets.

Comparative study on the physicochemical and functional properties of the mucilage in the carpel of Nymphaea odorata using ultrasonic and classical heating extractions

The cooked carpel of Nymphaea odorata has a large amount of transparent mucilage; however, the basic characteristics of this mucilage have not yet been reported. This study compared the physicochemical and functional properties of this mucilage obtained using conventional hot water extraction (HWM) and ultrasonic-assisted extraction (UAM). Neither HWM nor UAM affected the viability of mouse skin fibroblasts (NIH/3 T3) below 100 μg/mL. UAM had a higher yield production, phenol concentration, and in vitro antioxidant activity, but it had a lower viscosity and water-holding capacity than that of HWM. The Fourier transform infrared spectra revealed that the dialyzed HWM and UAM, named HWMD and UAMD, respectively, appeared to have major spectral differences at 1730 cm−1 and 1605 cm−1, implying that the degree of methylation was different between HWMD and UAMD. Compared to HWMD, UAMD in low-molecular weight polysaccharides increased. Indeed, the basic characteristics of native mucilage in the carpel of N. odorata were greatly changed by various extractions. Nevertheless, sugar analysis indicated that glucuronic acid was the main composition of HWMD and UAMD.

The immunomodulatory activities of licorice polysaccharides (Glycyrrhiza uralensis Fisch.) in CT 26 tumor-bearing mice

BackgroundThe increasing use of complementary and alternative medicine (CAM) has kindled the need for scientific evaluation of the mechanism of action of CAMs. Although, licorice, a common ingredient in many Traditional Chinese medicine (TCM) has attracted great attention for its antitumor and immunomodulatory activities, the mechanism of action of its polysaccharides is still unclear. Here we report the immunomodulatory activity of licorice polysaccharides in vivo.MethodsThe differential anticancer activities of licorice polysaccharides by tumorigenesis and immunomodulation was evaluated in vivo. Six weeks old, 120 CT-26 tumor bearing BALB/c mice, weighing 20 ± 2 g were used. They were randomly divided into six groups, three groups receiving high molecular weight (fraction A), low molecular weight (fraction B) polysaccharides and crude extract (fraction C); positive, negative and normal groups receiving cytoxin, saline and normal diet respectively. Weight of mice and tumors was determined and tumorigenicity assay calculated to determine the anticancer effects. Immunomodulatory potential was determined by immune organ indices, immune cell population and serum cytokine levels using immune organ weight and index, flow cytometry and cytokine/chemokine bead panel kit respectively.ResultsLicorice polysaccharides exhibited immunomodulatory activities in CT 26 tumor bearing BALB/c mice. The polysaccharides significantly suppressed tumor growth and increased immune organ index. Furthermore, the immunomodulatory effect was evident with activation of CD4+ and CD8+ immune cells population. The polysaccharides also affected the production of various cytokines, by increasing IL 2, IL 6, IL 7 levels and a decreasing TNFα levels.ConclusionIn summary, licorice polysaccharide especially of low molecular weight exhibit anticancer and immunomodulatory activities by suppressing tumor growth and improving general health of mice. They also augment the thymus/spleen index and population of T lymphocytes. Furthermore, the polysaccharides enhance the levels of serum antitumor cytokines, IL 2, IL 6 and IL 7 while decreasing pro-tumor cytokine TNFα.

Enhanced sludge solubilization and dewaterability by synergistic effects of nitrite and freezing

In this study, nitrite was added into sludge during freezing process to evaluate its role in waste activated sludge (WAS) solubilization and effect on sludge dewatering characteristics. The results showed that the introduction of 100 mg L−1 of nitrite could increase dissolved organic carbon (DOC) concentration from 29.5 to 48.8 mg DOC g−1 VSS under freezing conditions. More DOC was released with the increase of nitrite concentration. Freezing temperature, or freezing speed, also played a role in sludge solubilization. It was found that some readily-biodegradable low molecular weight (LMW) compounds, e.g. LMW protein, LMW polysaccharide, LMW neutrals, building blocks and LMW acids, were mainly released during the freezing process with the presence of nitrite. Interestingly, nitrite could also improve the sludge filterability at the lower nitrite concentration as a result of the increased sludge particle size. However, electrolytes (sodium nitrite) addition effects may mask such enhancement when nitrite concentration was high (800 mg L−1). The rheological characteristics of sludge could be well modeled by Herchel-Bulkley model and the introduction of nitrite into freezing process further increased sludge flowability and decreased sludge viscosity. These results indicated that freezing with the presence of suitable concentration of nitrite could promote sludge solubilization and dewaterability. As such, good liquid and solid separation can be achieved with the recovery of liquid stream as carbon source.

Succinoglycan Production Contributes to Acidic pH Tolerance in Sinorhizobium meliloti Rm1021.

In this work, the hypothesis that exopolysaccharide plays a role in the survival of Sinorhizobium meliloti at low pH levels is addressed. When S. meliloti was grown at pH 5.75, synthesis of succinoglycan increased, whereas synthesis of galactoglucan decreased. Succinoglycan that was isolated from cultures grown at low pH had a lower degree of polymerization relative to that which was isolated from cultures grown at neutral pH, suggesting that low-molecular weight (LMW) succinoglycan might play a role in adaptation to low pH. Mutants unable to produce succinoglycan or only able to produce high-molecular weight polysaccharide were found to be sensitive to low pH. However, strains unable to produce LMW polysaccharide were 10-fold more sensitive. In response to low pH, transcription of genes encoding proteins for succinoglycan, glycogen, and cyclic β(1-2) glucans biosynthesis increased, while those encoding proteins necessary for the biosynthesis of galactoglucan decreased. While changes in pH did not affect the production of glycogen or cyclic β(1-2) glucan, it was found that the inability to produce cyclic β(1-2) glucan did contribute to pH tolerance in the absence of succinoglycan. Finally, in addition to being sensitive to low pH, a strain carrying mutations in exoK and exsH, which encode the glycanases responsible for the cleavage of succinoglycan to LMW succinoglycan, exhibited a delay in nodulation and was uncompetitive for nodule occupancy. Taken together, the data suggest that the role for LMW succinoglycan in nodule development may be to enhance survival in the colonized curled root hair.

Influence of commercial inactivated yeast derivatives on the survival of probiotic bacterium Lactobacillus rhamnosus HN001 in an acidic environment

This study evaluated the influence of three inactivated yeast derivatives (IYDs) used in wine production, namely OptiRed®, OptiWhite® and Noblesse®, on the viability of the probiotic strain Lactobacillus rhamnosus HN001 in an acidic environment. Addition of the IYDs at 3 g/L significantly enhanced the survival of the probiotic bacteria by 2.75–4.05 log cycles after 10-h exposure in a pH 3.0 buffer. Acid stress assay with IYD components obtained after centrifugation and filtration revealed that water-soluble compounds were responsible for improving the acid tolerance of L. rhamnosus HN001 for all three preparations. Differences in protective effect amongst the IYDs on L. rhamnosus HN001 were observed when permeates and retentates of the water-soluble extracts, obtained through ultrafiltration with a 2 kDa membrane, were assayed against the lactic acid bacterium. Chemical analysis of the water-soluble components suggests that low molecular weight polysaccharides, specific free amino acids and/or antioxidants in the 2 kDa permeates could have contributed to the enhanced survival of L. rhamnosus HN001 during acid stress. The contrast amongst the 2 kDa retentates’ viability enhancing property may have been attributed to the differences in size and structure of the higher molecular weight carbohydrates and proteins, as the survival of the probiotic did not relate to the concentration of these compounds. These results suggests that oenological IYDs could potentially be applied to probiotic foods for enhancing the acid tolerance of the beneficial microorganisms, and consequently prolonging the shelf life of these products.

Longevity effect of a polysaccharide from Chlorophytum borivilianum on Caenorhabditis elegans and Saccharomyces cerevisiae.

The traditional Indian medicine, Ayurveda, provides insights and practical solutions towards a healthy life style. Rasayana is a branch of Ayurveda known for preserving and promoting health, enhancing the quality of life and delaying the aging process. In the traditional knowledge, the Rasayana herb, Chlorophytum borivilianum (C. borivilanum) is regarded as a general health promoting tonic that delays aging and increases lifespan, cognitive function and physical strength. Aging is a complex and multifactorial physiological phenomenon that manifests itself over a wide range of biological systems, tissues, and functions. Longevity is an obvious marker of physiological aging. Simple model systems such as the single-cell budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the nematode, Caenorhabditis elegans (C. elegans) are widely used to study the aging process and longevity. Here, we show that a polysaccharide fraction obtained from C. borivilianum increases the lifespan of S. cerevisiae and C. elegans, using an automated screening platform (ChronoscreenTM). Chemical analysis of this extract revealed a low molecular weight polysaccharide of 1000 Da, predominantly comprising Glu1→6Glu linkage. This polysaccharide showed significant dose-dependent extension of the median lifespan of S. cerevisiae by up to 41% and of the median lifespan of C. elegans by up to 10%. Taking cue from these results and the traditionally described benefits of Rasayanas on skin rejuvenation, we tested in vitro the polysaccharide for potential skin benefits. In a keratinocyte culture, we observed that this polysaccharide increased cell proliferation significantly, and induced synthesis of hyaluronic acid (HA), a well-known extracellular matrix component. Furthermore, when added to culture medium of human reconstructed epidermis, we observed an enhanced production of epidermal markers, e.g. CD44 and HA that are otherwise diminished in aged skin. Together, these results suggest that in addition to life-span extension of S. cerevisiae and C. elegans, a polysaccharide from the Rasayana herb, C. borivilianum may have beneficial effects on skin aging parameters.

Research Progress of Low Molecular Weight Polysaccharides

In this paper, lots of low molecular weight polysaccharides were reviewed. The advances of the extraction, purification, biological activity and structural changes of low molecular weight polysaccharides were detailed. And future research directions were outlooked, the article noted that future research should focus on the microscopic mechanism and establish appropriate physical and mathematical models. Low molecular weight polysaccharide can be applied to the new injection agent aspects of clinical anti-tumor and lowering blood pressure.

Aggregation and gelation of oat β-glucan in aqueous solution probed by NMR relaxometry

Nuclear magnetic resonance was used to study water proton transverse relaxation in oat β-glucan aqueous solutions during storage. The molecular weights of the samples ranged from 112 to 494kDa. The polysaccharide structure was identified by IR, GC–MS, NMR spectroscopy, and high performance anion exchange chromatography. In 1% solutions, 494kDa β-glucan formed stable solution phase. Aggregates were generated in the solution of low molecular weight polysaccharide. The transverse relaxation time of water associated with the aggregate microphase was about 15ms. The majority of the solution was bulk water, with a relaxation time of 800–1600ms. The proton exchange rate was about 103s−1, calculated from two-site exchange model. The proportion of β-glucan in the aggregate microphase increased from 0 to over 80% of the total β-glucan during storage of 112kDa samples. In the 4% solutions, the evolution of water proton relaxation associated with the gel network can be explained by the fractal structure theory. The relaxation behavior suggested the growth and syneresis of the gel network during the gelation of the low molecular weight β-glucan solutions.

Retention of Proanthocyanidin in Wine-like Solution Is Conferred by a Dynamic Interaction between Soluble and Insoluble Grape Cell Wall Components.

For better understanding of the factors that impact proanthocyanidin (PA) adsorption by insoluble cell walls or interaction with soluble cell wall-derived components, application of a commercial polygalacturonase enzyme preparation was investigated to modify grape cell wall structure. Soluble and insoluble cell wall material was isolated from the skin and mesocarp components of Vitis vinifera Shiraz grapes. It was observed that significant depolymerization of the insoluble grape cell wall occurred following enzyme application to both grape cell wall fractions, with increased solubilization of rhamnogalacturonan-enriched, low molecular weight polysaccharides. However, in the case of grape mesocarp, the solubilization of protein from cell walls (in buffer) was significant and increased only slightly by the enzyme treatment. Enzyme treatment significantly reduced the adsorption of PA by insoluble cell walls, but this effect was observed only when material solubilized from grape cell walls had been removed. The loss of PA through interaction with the soluble cell wall fraction was observed to be greater for mesocarp than skin cell walls. Subsequent experiments on the soluble mesocarp cell wall fraction confirmed a role for protein in the precipitation of PA. This identified a potential mechanism by which extracted grape PA may be lost from wine during vinification, as a precipitate with solubilized grape mesocarp proteins. Although protein was a minor component in terms of total concentration, losses of PA via precipitation with proteins were in the order of 50% of available PA. PA-induced precipitation could proceed until all protein was removed from solution and may account for the very low levels of residual protein observed in red wines. The results point to a dynamic interaction of grape insoluble and soluble components in modulating PA retention in wine.

The interaction of protamine nanocapsules with the intestinal epithelium: A mechanistic approach

Single-layer protamine and double layer polysialic acid (PSA)/protamine nanocapsules (NCs) were designed in order to be used as carriers to facilitate the transport of macromolecules across the intestinal epithelium. The rational for the design of these NCs was based on that protamine is a non-toxic yet potent cell-penetrating peptide, capable of translocating protein cargos through cell membranes, while PSA is a low molecular weight polysaccharide used to enhance the stability of macromolecules and nanocarriers. The aim of this work was to study in vitro the mechanism of interaction of these NCs with different intestinal cell models (Caco-2, Caco-2/Raji mimicking follicle associated epithelium and Caco-2/HT29-MTX to study the effect of mucus). For this, a fluorescent marker, TAMRA was covalently linked to protamine. The interaction and transport of the NCs with the Caco-2 cells was found to be concentration, temperature and size dependent. In all cases, the double layer PSA-protamine NCs exhibited a significantly higher transport compared to protamine NCs. On the other hand, the transport of the NCs was significantly higher in the co-culture (Caco-2/Raji monolayer) compared to the monoculture model (Caco-2 monolayer), implying that M cells are involved in the transport of these nanosystems. The formulations, administered intra-jejunally to healthy rats (4h fasting) resulted in a moderate reduction of the glucose levels (20% reduction), which lasted for up to 4h. This work raises prospects that protamine-based nanocapsules may have the potential as oral peptide delivery nanocarriers.

Low molecular weight carbohydrates released from Okara by enzymatic treatment under high hydrostatic pressure

Okara is an abundant, inexpensive and rich in dietary fibre by-product from soybean with potential prebiotic effect, although an increase in soluble dietary fibre (SDF) and low molecular weight carbohydrates (LMWC) would be desirable. Therefore, a simultaneous treatment with food-grade enzymes (Ultraflo® L or Viscozyme® L) and high hydrostatic pressure (HHP) was used to improve LMWC profile. Release of LMWC from Okara was monitored and estimated using a newly calibrated and validated HPLC method with an ion-exchange column and evaporative light scattering detection (HPLC-ELSD). Both enzymes improved the effect of the HHP treatment. Treatment at 600 MPa, 40 °C for 30 min achieved a decrease in the average molecular weight of polysaccharides from 12.74 ± 3.97 kDa to 3.85 ± 1.33 or 1.59 ± 0.40 kDa when Ultraflo® L or Viscozyme® L were used, respectively. Ultraflo® L (15 min) and Viscozyme® L (30 min) at 600 MPa, were the most effective treatments. Using industrial by-products as a source of nutraceuticals is interesting from an economical point of view. The content of low molecular weight polysaccharides of Okara has been maximized to increase its value as a prebiotic. An industrial adapted treatment with high hydrostatic pressure assisted by food-grade enzymes has been employed. In addition, a new HPLC-ELDS method has been calibrated for monitorizating the process.

Preparation and inhibition on α-d-glucosidase of low molecular weight polysaccharide from Cordyceps militaris

The structural properties and the inhibition on α-d-glucosidase activity of the low molecular weight (LCMPs-II) obtained from the optimized acid hydrolysis of the Cordyceps militaris polysaccharides (CMPs) were investigated in this paper. The LCMPs-II with a molecular weight of 28 KDa mainly composed of rhamnose, xylose and glucose with the molar ratio of 1: 2.19: 6.73 was separated from LCMPs-I which was the acid hydrolysis product of CMPs by chromatography on Sephadex G-100 column. The solubility of LCMPs-II was tested to be 32.12±1.05g in 100mL distilled water under 25°C. Its solubility was almost as twice as that of CMPs. Afterward, the structural features of LCMPs-II was investigated by a combination of chemical and instrumental analysis such as the specific rotation determination, FT-IR, periodate oxidation-Smith degradation, Congo-red, GC, scanning electron microscope and NMR. The results showed that the optical rotation of LCMPs-II was +25° and it was 1,3-branched-rhamnoxyloglucan which had a linear backbone of (1→4)-linked α-d-glucopyranose (α-d-Glcp units).

Rapid preparation of functional polysaccharides from Pyropia yezoensis by microwave-assistant rapid enzyme digest system

This study describes a simple preparation of functional polysaccharides from Pyropia yezoensis using a microwave-assistant rapid enzyme digest system (MAREDS) with various carbohydrases, and evaluates their antioxidative effects. Polysaccharide hydrolysates were prepared using MAREDS under different hydrolytic conditions of the carbohydrases and microwave powers. Polysaccharides less than 10kDa (Low molecular weight polysaccharides, LMWP, ≤10kDa) were efficiently obtained using an ultrafiltration (molecular weight cut–off of 10kDa). MAREDS increases AMG activation via an increased degree of hydrolysis; the best AMG hydrolysate was prepared using a 10:1 ratio of substrate to enzyme for 2h in MAREDS with 400W. LMWP consisted of galactose (27.3%), glucose (64.5%), and mannose (8.3%) from the AMG hydrolysate had stronger antioxidant effects than the high molecular weight polysaccharides (>10kDa). We rapidly prepared functional LMWPs by using MAREDS with carbohydrases, and suggest that LMWP might be potentially a valuable algal polysaccharide antioxidant.

Carboxymethylated degraded polysaccharides from Enteromorpha prolifera: Preparation and in vitro antioxidant activity

In order to improve the bioactivities of the polysaccharide from Enteromorpha prolifera (PE), crude PE (Mw 1400kDa) was degraded to low molecular weight polysaccharide (44kDa) in the presence of hydrogen peroxide/ascorbic acid, followed by carboxymethylation. The reaction conditions for carboxymethylation of degraded polysaccharide (DPE) were optimized by Response Surface Methodology. The carboxymethyled degraded polysaccharide (CDPE) obtained under optimized conditions, with a degree of carboxymethylation of 0.849, was characterized by FT-IR and 13C NMR. The molecular weight of CDPE was measured to be 53.7kDa. CDPE was evaluated for its antioxidant activity by determining the ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and superoxide anion radicals, and by determining the ferric reducing power. The antioxidant activity of CDPE was found to be greatly improved in comparison with degraded polysaccharide (DPE) and crude polysaccharide from Enteromorpha prolifera (PE).

Physiological Degradation of Pectin in Papaya Cell Walls: Release of Long Chains Galacturonans Derived from Insoluble Fractions during Postharvest Fruit Ripening.

Papaya (Carica papaya L.) is a fleshy fruit that presents a rapid pulp softening during ripening. However, the timeline on how papaya pectinases act in polysaccharide solubilization and the consequent modification of the cell wall fractions during ripening is still not clear. In this work, the gene expression correlations between, on one hand, 16 enzymes potentially acting during papaya cell wall disassembling and, on the other hand, the monosaccharide composition of cell wall fractions during papaya ripening were evaluated. In order to explain differences in the ripening of papaya samplings, the molecular mass distribution of polysaccharides from water-soluble and oxalate-soluble fractions (WSF and OSF, respectively), as well as the oligosaccharide profiling from the WSF fraction, were evaluated by high performance size exclusion chromatography coupled to a refractive index detector and high performance anion-exchange chromatography coupled to pulse amperometric detection analyses, respectively. Results showed that up-regulated polygalacturonase and β-galactosidase genes were positively correlated with some monosaccharide profiles. In addition, an overall increase in the retention time of high molecular weight (HMW) and low molecular weight (LMW) polysaccharides in WSF and OSF was shown. The apparent disappearance of one HMW peak of the OSF may result from the conversion of pectin that were crosslinked with calcium into more soluble forms through the action of PGs, which would increase the solubilization of polysaccharides by lowering their molecular weight. Thus, the results allowed us to propose a detailed process of papaya cell wall disassembling that would affect sensorial properties and post-harvesting losses of this commercially important fruit.

In vitro anticancer activity of the laminarans from Far Eastern brown seaweeds and their sulfated derivatives

Brown seaweeds have drawn worldwide attention due to their involvement in many industrial applications. They produce neutral low molecular weight polysaccharides (laminarans) with beneficial biological activities. However, the anticancer activity and molecular mechanism of the laminarans and their sulfated derivatives have not been investigated in detail. Herein, the laminarans from brown seaweeds Saccharina cichorioides, Saccharina japonica, and Fucus evanescens were isolated. The laminarans from S. cichorioides and S. japonica were confirmed to contain a main chain of β-(1→3)-D-glucopyranose with single branches at C6. The laminaran from F. evanescens consisted of not only β-(1→3)-linked D- glucopyranose but also includes single β-(1→6)-linked D-glucose residues. The branches at C6 are presented as glucose or as gentiobiose. The sulfated laminarans with different degrees of sulfation were obtained by the chlorosulfonic acid-pyridine assay. In modified polysaccharides, the positions of sulfates are directly predetermined by the structure of native laminarans. The laminarans and their sulfated derivatives inhibited proliferation, colony formation, and migration of human colorectal adenocarcinoma, melanoma, and breast adenocarcinoma cells in different manners. The sulfated laminaran from F. evanescens possessed the highest anticancer activity in vitro and effectively prevented migration of breast adenocarcinoma cells by inhibiting of the Matrix Metalloproteinases 2 and 9 activity.

Structural characterization and anti-inflammatory activity of two novel polysaccharides from the sea squirt, Ascidiella aspersa

It is now recognized that certain polysaccharides can exhibit anti-inflammatory activity, including the glycosaminoglycan (GAG) heparin that is widely used as an anti-coagulant drug. However, it would be desirable to identify molecules that retain the anti-inflammatory actions of heparin, but that are devoid of significant anti-coagulant activity. In the present study we have identified a number of novel GAG and GAG-like polysaccharides (VRP327) from marine organisms, most of which were resistant to digestion by heparinase II and chondroitinase ABC. Fourier transform infra-red spectrum (FTIR) revealed species with variable degrees of sulphation and monosaccharide analysis revealed a range of sugar compounds, which in some cases included sugars not present in mammalian GAGs. 1H NMR spectra of these species are consistent with the structures of complex polysaccharides. From an initial screening cascade to remove compounds having significant anti-coagulant activity and no overt cytotoxicity, we identified a high molecular weight oversulphated dermatan sulphate (VRP327) isolated from the tunicate Ascidiella aspersa which was fully characterised by NMR spectroscopy. This material was depolymerised to produce well characterized low molecular weight fractions which were demonstrated to be non-toxic, with low levels of anti-coagulant activity, and to have demonstrable anti-inflammatory activity assessed in several in vitro and in vivo models. The identification of low molecular weight polysaccharides having significant anti-inflammatory activity without significant anti-coagulant activity may provide novel templates for the development of a novel class of anti-inflammatory drugs.

Structural characterization of low molecular weight polysaccharide from Astragalus membranaceus and its immunologic enhancement in recombinant protein vaccine against systemic candidiasis

Structure and immunologic enhancement of low molecular weight polysaccharide (LMW-ASP) isolated from the root of Astragalus membranaceus (Fisch) Bge. Were detected in recombinant protein vaccine. Structure analysis of LMW-ASP revealed that LMW-ASP (Mw=5.6kDa) was an acid heteropolysaccharide, which consisted of Glc, Gal, Ara, Xyl and GalA in ratio of 10.0：1.3：1.7：1.0：0.9. Recombinant protein (rP-HSP90C) contained epitope C (LKVIRK) from heat shock protein 90 (HSP90) of Candida albicans was used as a vaccine. The results indicated that LMW-ASP significantly promoted specific antibody titers IgG, IgG1, IgG2b, and IL-2, IL-4, IL-10, IL-12 in sera of mice immunized with rP-HSP90C (p<0.05). It was also found LMW-ASP improved DTH response in HSP90C-injceted mice. More importantly, the mice immunized with rP-HSP90C/LMW-ASP had fewer CFU (colony forming unites) in the kidneys compared to the mice immunized with rP-HSP90C (p<0.05). Therefore, LMW-ASP could be exploited into the novel adjuvant to enhance the efficacy of recombinant protein vaccine.