Fractionation and characterization of chitosan by analytical SEC and H NMR after semi-preparative SEC

Heterogeneity in molecular weight and degree of deacetylation (DDA) of chitosans from different sources and preparation methods were studied by fractionating chitosans, using semi-preparative SEC, and then determining molecular weight profiles of fractions by analytical SEC with multi-angle laser light scattering (SEC–MALLS), and degree of deacetylation (DDA) by ¹H NMR. Fractionation of two high molecular weight chitosans from different manufacturers, produced fractions that spanned a wide range of molecular weight (number-average M_n), from 65 to 400 kDa in one case, that was not evident when unfractionated material was directly analyzed by SEC providing M_n = 188 kDa and PDI = M_w/M_n = 1.73. In a second case, fractions ranged from 20 to 600 kDa with unfractionated M_n = 145 kDa and PDI = 1.83. Fractionation of low molecular weight chitosans also showed a broad range of molecular weight in the original material, however, the fractions obtained with the TSKgel G4000W column in the M_n range of 5–100 kDa were essentially monodisperse with PDIs between 1.0 and 1.4. The DDA of one low molecular weight chitosan (10 kDa) produced by nitrous acid degradation was dependent on the M_n of the fraction. This semi-preparative fractionation procedure revealed important compositional heterogeneities of chitosans not evident in unfractionated material, and permitted the production of monodisperse low molecular weight chitosans with homogeneous properties.     

Characterization of galactomannans isolated from legume endosperms of Caesalpinioideae and Faboideae subfamilies by multidetection aqueous SEC

Galactomannans isolated from legume seed endosperms, including those of commercial interest, have been characterized by multidetection aqueous SEC. Galactomannans derived from seeds of the Faboideae subfamily had substantially higher M_w than those from Caesalpinioideae seeds (M_w,Fab = 2.4–3.1 × 10⁶ g/mol, M_w,Caes. = 0.86–2.1 × 10⁶ g/mol) and within the latter botanical subfamily, an apparent correlation between M_w and the degree of galactose substitution DG was found. The molar mass distributions were unimodal and differed primarily by a scale factor, with distributional widths narrower than a true Flory ‘most-probable distribution’; good fits to Schulz–Zimm model were obtained. Across subfamilies no differences were found in the exponents of [η]–M and R_v–M relationships (0.61 ± 0.02, 0.54 ± 0.01, respectively), the Flory chain stiffness ratio (C_∞ = 20 ± 1 (BSF analysis)), or the persistence length (L_p = 5.5 ± 0.2 nm) obtained from SEC fraction data. However, it was found that prefactors in the [η]–M and R_v–M relationships as well as the unperturbed parameter K_Θ decrease in proportion to DG and therefore chain density. Generalized relationships incorporating galactose-dependent prefactors were therefore developed to model SEC fraction data of native galactomannans ([η]_GM = (1800 ± 200) × M_o^−1.61 × M^0.61±0.02, R_v,GM = 0.63 ± 0.05 × M_o^−0.54 × M^0.54±0.01) as well as lower-M fractions obtained by ultrasonication ([η]_GM = (730 ± 100) × M_o^−1.71 × M_w^0.71±0.02, R_v,GM = 0.49 ± 0.05 × M_o^−0.57 × M_w^0.57±0.01, M ≈ 1 × 10⁵-native). As a consequence of this dependence and the observed patterns in molar mass variation, [η] varies within a narrow range for galactomannans as a whole despite substantial M_w differences.     

Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

Effect of microwave irradiation on the molecular and structural properties of hyaluronan

Hyaluronan (Na⁺ salt of hyaluronic acid, HA) was extensively depolymerised by HCl-catalyzed hydrolysis at pH 3 for up to 500 min under temperature-controlled microwave irradiation. The effects of microwave heating on the hydrodynamic properties of the polysaccharide were determined by SEC-MALLS and viscometry. The weight-average molecular mass (M_w) of HA decreased from 1.44 × 10⁶ to 5000, reaching the region of higher oligosaccharides. The scission of HA chains was found to proceed randomly during the whole degradation process. Treatment of the M_w and intrinsic viscosity data according to the Mark–Houwink equation, [η] = k × M_w^α suggested three relationships with α₁ = 0.46 for M_w > 500,000, α₂ = 0.84 for M_w between 500,000 and 50,000, and α₃ = 1.13 for M_w < 50,000. The results revealed that HA with M_w > 10,000 adopts a stiffish coil conformation in solution. As monitored by FT-IR and NMR spectroscopic techniques, the primary structure of the HA chains was maintained during the microwave-assisted hydrolysis at pH 3 at 105 °C. At reaction times larger than 240 min, uv spectroscopy suggested the depolymerisation of HA was accompanied by formation of by-products produced by side reaction.     

Molecular mass and chain conformations of Rhizoma Panacis Japonici polysaccharides

The molecular mass and size of five water-soluble polysaccharides isolated from Rhizoma Panacis Japonici (RPJ) were determined with laser light scattering (LLS), size-exclusion chromatography (SEC) combined with LLS (SEC–LLS), dynamic light scattering (DLS), as well as transmission electron microscope (TEM). Their weight-average molecular masses (M_w) were 3.5 × 10⁴, 1.47 × 10⁵, 1.24 × 10⁶, 9.26 × 10⁵ and 1.36 × 10⁶, radii of gyration (<s²>_z^1/2) were 14.7, 31.7, 50.8, 41.8 and 40.4 nm, and hydrodynamic radii (R_h) were 19.9, 37.5, 66.2, 52.1, and 55.2 nm, respectively. The results showed that molecular masses and sizes of the polysaccharides were influenced by the pH and temperature of the extraction mediums. The conformation parameters were calculated from the above data according to polymer solution theory. The values of ρ (= <s²>_z^1/2/R_h) were from 0.7 to 0.8, exponents (ν) of <s²>_z^1/2 = k M_w^ν were from 0.31 to 0.43, and fractal dimension (d_f) were from 2.3 to 3.2, respectively. The results revealed that all of the polysaccharides existed as spheres in 0.15 M NaCl aqueous solution. TEM and atomic force microscope (AFM) further confirmed the spherical morphologies of these molecules. The spherical conformations of the polysaccharides were a result of their highly branched structures.     

Rheological properties of concentrated solutions of gellan in an ionic liquid

The rheological properties of solutions of gellan were examined at high concentrations where there is entanglement coupling between gellan chains. An ionic liquid 1-butyl-3-methylimidazolium chloride (BmimCl) was used as a solvent. Concentrated solutions of gellan in BmimCl were obtained by using a hot-molding technique. The concentration of gellan was varied from 1.9 × 10² to 4.8 × 10² kg m⁻³. The measurement temperature ranged from 50 to 100 °C. The master curve of the angular frequency dispersion of the storage modulus for the gellan solutions showed a rubbery plateau at high angular frequency. The molecular weight between entanglements (M_e) for gellan was obtained from the plateau modulus. From the concentration dependence curve of M_e, M_e for gellan in the molten state was determined to be 2.3 × 10³.     

Chain conformation and anti-tumor activities of phosphorylated (1→3)-β-d-glucan from Poria cocos

(1→3)-β-d-Glucan isolated from Poria cocos was phosphorylated to obtain a series of phosphorylated derivatives. Their structures, weight-average molecular weights (M_w), and chain conformation were studied by ¹³C NMR, ³¹P NMR, static laser light scattering and viscometry. The experimental results revealed that the phosphorylated glucan existed as relatively extended flexible chain in 0.15 M NaCl aqueous solution, and exhibited relatively strong inhibition against S-180 tumor cell in vitro and in vivo. In vivo, the fractions with relatively high molecular weight at low dosage exhibited stronger anti-tumor activities. The results revealed that the molecular weights and molecular conformation could influence the anti-tumor activities. The molecular weight ranging from 2.6 × 10⁴ to 26.8 × 10⁴ and the extended chain conformation were beneficial to enhance the anti-tumor activity, as a result of the increasing of the interaction between polysaccharide and immune system.     

Rapid method to determine the molecular weight of dextrins and dextrans

A rapid method was developed to determine the molecular weight (M_n) of β-limit dextrin and dextrans (Leuconostoc mesenteroides) using a reducing power approach. The M_n of the β-limit dextrin was also estimated from high performance liquid chromatography (HPLC). Chromatograms were pre-calibrated with the dextrans. The three dextrins had a M_n of 2.09, 2.40 and 2.63 × 10⁵ using the reducing method and 4.80, 5.90 and 2.80 × 10⁵ by HPLC. The method could be employed to estimate M_n of dextrins where chromatographic systems were not available.     

Physical properties of fungal chitosan

Fungi are promising alternative sources of chitosan. This study evaluated the physical properties of fungal chitosan from Absidia coerulea (AF 93105), Mucor rouxii (Ag 92033), and Rhizopus oryzae (Ag 92033). FT-IR and X-ray diffraction of the extracted products showed typical chitosan peak distributions which confirmed the extracted products to be chitosan. All of their glucosamine contents and degrees of deacetylation (DD) were over 80%, not showing obvious differences respectively. However, differences had been observed in their molecular weight (M_w), ranging from 6.6  to 560 kDa. The results of this study demonstrated that different fungi could produce different M_w chitosan with high DD and high purity.     

Physicochemical properties and antitumor activities for sulfated derivatives of lentinan

Five fractions of lentinan, a β-(1→3)-d-glucan bearing β-(1→6)-d-glucopyranosyl branches, were treated with chlorosulfonic acid for 90 min at 60 °C in pyridine medium to synthesize water-soluble sulfated derivatives having the substitution degree of 1.44–1.76. The ¹³C NMR spectra of the sulfated β-glucans indicated that the C-6 position was preferentially substituted by the sulfate groups. The values of the weight-average molecular weight (M_w), radius of gyration (), and intrinsic viscosity ([η]) of the sulfated lentinan fractions were determined by size-exclusion chromatography with multi-angle laser light scattering (SEC–MALLS) and viscometry in 0.15 M aq NaCl at 25 °C, respectively. The dependence of [η] on M_w for the sulfated lentinan was found to be [η] = 8.93 × 10⁻³ (mL/g) in 0.15 M aq NaCl (for M_w ranging from 14.6 × 10⁴ to 50.4 × 10⁴). On the basis of the Yamakawa–Fujii–Yoshizaki (YFY) theory, the conformational parameters of the sulfated lentinan were calculated as 950 nm⁻¹ for the molar mass per unit contour length (M_L), 4.8 nm for the persistence length (q), and 13.9 for the characteristic ratio (C_∞), indicating relatively extended single flexible chains in solution. The sulfated glucan fractions exhibited in vitro antiproliferative activities against sarcoma 180 (S-180) cells, and their inhibition ratios were lower than that of the triple-helix lentinan, but higher than that for the one with single random-coil lentinan chains.     

Effect of molecular weight of chitosan on antimicrobial properties and tissue compatibility of chitosan-impregnated bacterial cellulose films

Bacterial cellulose-chitosan (BC-C) films were developed by immersing purified BC pellicles in 1.5 ~ 2.0% (w/v) acetic acid solutions containing chitosan of varying molecular weights. Effects of different molecular weight of chitosan on physical, biological and antimicrobial properties of the composite films were investigated. The cumulative chitosan absorption capacities with M_w of 141,000, 199,000, and 263,000 were 38.43, 24.65, and 23.89 mg/cm³ of dry BC film, respectively. The cumulative release profiles of chitosan from the films strongly depended on molecular weight of chitosan and pH of solution. The order of release of chitosan from the BC-C films was dependent on molecular weight as follows: M_w 141,000 > M_w 199,000 > M_w 263,000. All BC-C films showed the antimicrobial abilities against Staphylococcus aureus and Aspergillus niger but had no inhibitory effect on the growth of Escherichia coli. The BC-C films supported for adhesion, spreading and proliferation of both human skin keratinocytes and fibroblasts. The antibacterial activity against S. aureus of the BC-C with the highest Mw chitosan (263,000) was higher than those of the others. On the other hand, the BC-C films with the lowest M_w chitosan (141,000) promoted the growth of human skin cells more than those of the others.     

Molecular mass and antitumor activities of sulfated derivatives of α-glucan from Poria cocos mycelia

Two kinds of water-insoluble (1 → 3)-α-d-glucan samples, ab-PCM3-I and ac-PCM3-I, isolated from different Poria cocos mycelia were sulfated, to produce two series of water-soluble derivatives ab-PCM3-I-S1–S5 and ac-PCM3-I-S1–S5, respectively. The derivatives having different weight-average molecular mass (M_w) were produced by changing reaction temperature and time as well as molar ratios between chlorosulfonic acid and number of hydroxyl groups in the glucan. The degrees of substitution (DS) of the sulfated derivatives were analyzed by elemental analysis (EA) to be 0.39–0.67 for ab-PCM3-I-S and 0.73–0.96 for ac-PCM3-I-S, respectively. The M_w and the intrinsic viscosity ([η]) of the samples ab-PCM3-I-S and the ac-PCM3-I-S were measured by size exclusion chromatography combined with laser light scattering (SEC–LLS) and viscometry in phosphate buffer solution (PBS) at 37 °C. The results indicated that their M_w ranged from 2.0 to 11.3 × 10⁴ for the samples ab-PCM3-I-S, and 4.7 to 40.0 × 10⁴ for the samples ac-PCM3-I-S. Moreover, the antitumor activities of the sulfated derivatives ab-PCM3-I-S and ac-PCM3-I-S against Sarcoma 180 tumor cell tested both in vitro and in vivo are significantly higher than those of the native α-d-glucans. Therefore, a moderate range of molecular mass from 2.0 × 10⁴ to 40.0 × 10⁴, relatively high chain stiffness and good water solubility of the sulfated derivatives are beneficial to the enhancement of their antitumor activities.     

PEGylation of rhIL-1RA increased its solution stability at room temperature

Recombinant human interleukin-1 receptor antagonist (rhIL-1RA) is an important cytokine in the treatment of inflammatory diseases. However, it is instable in aqueous solution and prone to degrade without the addition of any excipient. Following the 30- or 60-day storage in 50 mM sodium acetate (pH 5.0) at room temperature, rhIL-1RA markedly degraded into three species (denoted as P1, P2 and P3 in this study), the bioactivities of which to a different extent was lost (from 9.72 × 10⁴ UI/mg to 3.07 × 10³ UI/mg for P1, 5.49 × 10³ UI/mg for P2, 1.09 × 10⁴ UI/mg for P3, respectively). To solve this problem, we prepared the mono-PEGylated rhIL-1RA with propionaldehyde mPEG (ALD-PEG, M_w 5000 Da). The conjugate showed more favorable stability than original protein, and remained homogeneous under the similar storage conditions. In addition, the activity of the conjugate was well retained (from 5.80 × 10⁴ UI/mg to 4.92 × 10⁴ UI/mg), compared to that of original protein. The results based on the combination analysis of CD, ion exchange chromatography and RP-HPLC, revealed that the stability improvement of rhIL-1RA majorly benefited from the PEG strands protection against the protein conformational changes occurred during the storage.     

An influence of methane concentration on the methanotrophic activity of a model landfill cover

Evaluation of kinetic parameters of methane oxidation under various conditions, on the basis of an analysis of the literature and the authors’ own laboratory research, is presented. Variation in methanotrophic activity in the profile of a simulated landfill cover was observed. The greatest activity was found at a depth of 60 cm. A low affinity (1/K_M) and high potential activity (V_max) were observed. V_max values ranged from 0.11 × 10⁻³ to 0.86 × 10⁻³ units. The values of K_M ranged from 0.6 to 2.9% of CH₄ (v/v).     

Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking

These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C₁-BODIPY-C₁₂ in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis–Menten kinetics; the apparent efficiency (k_cat/K_T) of this process increases over 2-fold (2.1 × 10⁶–4.5 × 10⁶ s⁻¹ M⁻¹) upon adipocyte differentiation. The V_max values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 × 10⁶ s⁻¹ M⁻¹ versus 1.5 × 10⁶ s⁻¹ M⁻¹). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V_max values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The same cells had reduced efficiency for fatty acid transport (ranging from 0.82 × 10⁶ s⁻¹ M⁻¹ to 1.35 × 10⁶ s⁻¹ M⁻¹).     

Iron plaque formation on wetland plants and its influence on phosphorus,calcium and metal uptake

We investigated Fe plaque formation and Ca, Cu, Mn, Zn, and P uptake capacities of fifteen kinds of wetland plants. The test plants were cultured in 3 l nutrient solutions for 8 days. Fe plaque was induced by adding 200 mg l⁻¹ Fe²⁺ as FeSO₄·7H₂O for 4 days in one set of experiment and 8 days in another. This plaque ranged from 2.38 to 8.67 mg g⁻¹ of plant root after 4 days and from 4.56 to 15.71 mg g⁻¹ of plant root after 8-day treatment. In both experimental durations, the plaque was significantly correlated with root surface area (r = 0.904 and 0.878, P < 0.01). Thus, Canna generalis, Typha latifolia and Thalia dealbata, with their larger root surface areas (>1,400 cm²), formed relatively greater Fe plaque amounts. The amounts of Ca, Cu, Zn and P in the Fe plaques were significantly correlated with Fe plaque amount, (r = 0.819, 0.742, 0.693, 0.917, respectively, for these four elements for the 4-day treatment; and r = 0.917, 0.768, 0.949, 0.872, respectively, for 8-day treatment, P < 0.01). Plants varied widely in accumulating Ca, Cu, Mn, Zn, and P in their tissues. The amounts accumulated on root were significantly correlated with Fe plaque amount in both for 4- and 8-day exposure treatments with Fe (r = 0.973, 0.847, 0.709, 0.837, 0.892, respectively, for 4-day treatment; and r = 0.943, 0.691, 0.843, 0.957, 0.983, respectively, for 8-day treatment, P < 0.01). No such significant correlations were found for the Fe plaque in shoot. Canna generalis, Typha latifolia and Thalia dealbata were superior in Ca, P and Zn uptake, while Canna generalis and Thalia dealbata accumulated Cu and Mn well in case of concentrated wastewater treatment.     

Kinetic analyses for bindings of concanavalin A to dispersed and condensed mannose surfaces on a quartz crystal microbalance

A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K_a) and binding and dissociation rate constants (k_on and k_off) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K_a = (4 ± 1) × 10⁶ M⁻¹, k_on = (4 ± 1) × 10⁴ M⁻¹ s⁻¹, and k_off = (12 ± 2) × 10^–3 s⁻¹. On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K_a = (14 ± 2) × 10⁶ M⁻¹, k_on = (14 ± 2) × 10⁴ M⁻¹ s⁻¹, and k_off = (7 ± 2) × 10^–3 s⁻¹. Thus, K_a for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k_on) and a three times smaller dissociation rate constant (k_off). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface.     

Antioxidant reactivity toward nitroxide probes anchored into human serum albumin. A new model for studying antioxidant repairing capacity of protein radicals

A new strategy to evaluate accessibility of antioxidants to radical proteins has been developed using nitroxide prefluorescent probes anchored into human serum albumin (HSA). Binding association constants for the nitroxide probes C₃₄₃T and QT with HSA were 5 × 10⁴ and 9 × 10⁴ M⁻¹, respectively. Rate constants for the nitroxide reduction by antioxidants in HSA were determined finding k_HSA/k_buffer ratio of 0.8, 1.9, and 0.075 for ascorbic acid, Trolox, and caffeic acid, respectively, for the nitroxide C₃₄₃T reduction.     

In vitro cytogenetic effects of Andrographis paniculata (kalmegh) on arsenic

In vitro effects of arsenic in human peripheral lymphocytes (HPL) at three different doses – 3.6×10⁻⁴, 1.4×10⁻³ and 0.72×10⁻³ μM for 24 h before harvesting on sister chromatid exchanges (SCE), Cell cycle proliferative index/replicative index (CCPI/RI), %M₁, %M₂ and %M₃, population doubling time (PDT) and average generation time (AGT) were examined. Andrographis paniculata (commonly referred to as ‘kalmegh’) has been used for centuries in traditional Indian and Chinese herbal medicine as a safe, natural folk remedy for assorted health concerns. In the present study, kalmegh (0.01 μg/7 ml culture media) was used along with the highest dose of arsenic; the results showed that arsenic induced increase in these genotoxic endpoints were fairly diminished by kalmegh. In addition, mutagenic in vitro effect of ethyl methanesulphonate (EMS) was used as a positive control in this study. It is thus concluded from this study that Andrographis has a protective role in arsenic toxicity.     

Aluminum stress on sorghum growth and nutrient relationships

Summary Sorghum plants were grown in the greenhouse in modified Steinberg nutrient solution containing ten Al rates (0 to 297 μM) and harvested 28 days after transplanting. Top and root dry weight were not affected by added Al up to 74 μM; but decreased sharply at concentration of 148 μM and greater. Aluminum concentrations in blade 1 (recently matured blade) and plants remained constant from 0 to 297 μM added Al. Root Al concentration increased as added Al increased. No correlation existed between top dry weight and Al concentration in blade 1 or in plant. Root Al concentration was related to top dry weight and root dry weight to estimate the Al critical toxicity level. The Al critical toxicity levle in the root was 54 mmol kg⁻¹ root dry weight basis for either top or root dry weight. In blade 1 Cu concentration negatively correlated with Al while Fe and P were positively correlated. In roots Ca, Mg, Mn and Fe concentrations were negatively correlated with Al while Zn, Cu, P, and K were positively correlated with Al concentration.