Radical Depolymerization Research Articles

Depolymerization of polysaccharides from Opuntia ficus indica: Antioxidant and antiglycated activities

The extraction, purification and degradation of polysaccharides from Opuntia ficus indica cladodes, as well as the evaluation of their antioxidant and antiglycated activities in vitro were investigated. The optimization of the extraction showed that extraction by ultrasound at 40°C presented the best carbohydrates yield. The degradation of the extracted polysaccharides was achieved by free radical depolymerization with H2O2 in the presence of copper(II) acetate for various reaction times. Sugar contents were determined by colorimetric assays. The macromolecular characteristics of the different isolated and degraded carbohydrates were carried by size exclusion chromatography (SEC/MALS/VD/DRI). These experiments showed that all samples are polysaccharides, which are probably pectins and that molecular weight (Mw) has decreased from 6,800,000 to 14,000g/mol after 3h of depolymerization without changing the structure. Preliminary antioxidant and antiglycated tests indicated that degraded polysaccharides for 2 and 3h showed even better antioxidant and antiglycated activities.

Profiling pneumococcal type 3-derived oligosaccharides by high resolution liquid chromatography–tandem mass spectrometry

Pneumococcal type-3 polysaccharide (Pn3P) is considered a major target for the development of a human vaccine to protect against Streptococcus pneumoniae infection. Thus, it is critical to develop methods for the preparation and analysis of Pn3P-derived oligosaccharides to better understand its immunological properties. In this paper, we profile oligosaccharides, generated by the free radical depolymerization of Pn3P, using liquid chromatography (LC)–tandem mass spectrometry (MS/MS). Hydrophilic liquid interaction chromatography (HILIC)–mass spectrometry (MS) revealed a series of oligosaccharides with an even- and odd-number of saccharide residues, ranging from monosaccharide, degree of polymerization (dp1) to large oligosaccharides up to dp 20, generated by free radical depolymerization. Isomers of oligosaccharides with an even number of sugar residues were easily separated on a HILIC column, and their sequences could be distinguished by comparing MS/MS of these oligosaccharides and their reduced alditols. Fluorescent labeling with 2-aminoacridone (AMAC) followed by reversed phase (RP)-LC–MS/MS was applied to analyze and sequence poorly separated product mixtures, as RP-LC affords higher resolution of AMAC-labeled oligosaccharides than does HILIC-based separation. The present methodology can be potentially applied to profiling other capsular polysaccharides.

Influence of chemical and structural features of low molecular weight heparins (LMWHs) on skin penetration

Low molecular weight heparins (LMWHs) are obtained from unfractionated heparin (UFH) through different depolymerization methods (DM), which produce compounds having specific chemical features and biological activity. It is then supposed that LMWHs also exhibit different skin permeability properties. The current work aimed to get an insight on the in vitro passive diffusion through human epidermis of six commercially available LMWHs in comparison with UFH. The in vitro studies were performed using Franz diffusion cells. Heparins samples were assayed measuring the anti-factor Xa activity. Circular dichroism was used to evaluate the effect of the counter-ion (sodium or calcium) on the chain flexibility. The penetrated amounts after 24h (Q24) of sodium LMWHs were related to Mw by an exponential relationship (R=−0.758). The flux resulted dependent by DM following the rank order: β-elimination (8–11mIU/cm2h range)>deaminative cleavage (5–7mIU/cm2h range)>radical depolymerization (0.1mIU/cm2h). Finally, the calcium ion, reducing the chain flexibility, significantly affected the Q24 (0.001±0.000 and 0.157±0.049IU/cm2 for calcium and sodium nadroparin, respectively). Both the lower Mw and the introduction of new residues at the chain ends improved the skin penetration of LMWHs with respect to UFH (Q24=0.001±0.001IU/cm2), with bemiparin and enoxaparin being the most interesting compounds.

Structural characterization and antioxidant activities of κ-carrageenan oligosaccharides degraded by different methods

In the present study, four kinds of κ-carrageenan oligosaccharides were obtained by the degradation of parent κ-carrageenan using free radical depolymerization, mild acid hydrolysis, κ-carrageenase digestion and partial reductive hydrolysis, respectively. Their structure types were accurately and comparatively elucidated by ESI-MS and CID MS/MS. The antioxidant activities of different degraded products were investigated by four different antioxidant assays, including superoxide radical scavenging activity, hydroxyl radical scavenging activity, reducing power and DPPH radical scavenging activity. The methods of depolymerization had an influence on the antioxidant activities of κ-carrageenan oligosaccharides obtained from κ-carrageenan. These results indicated that the antioxidant activities of κ-carrageenan oligosaccharides could be related to the degree of polymerization, the content of reducing sugar and sulfate groups, and the structure of reducing terminus.

Method to Detect Contaminants in Heparin Using Radical Depolymerization and Liquid Chromatography–Mass Spectrometry

Heparin is a critically important anticoagulant drug that was contaminated with a persulfonated polysaccharide in 2008, resulting in a number of severe adverse reactions, some leading to death. Controversy remains as to the precise composition of the 2008 contaminant, and new information suggests that heparin may now be subject to adulteration with a new, difficult to detect, contaminant, N-sulfo oversulfated chondroitin sulfate. This study synthesizes this new potential contaminant and describes the use of radical depolymerization followed by liquid chromatography-mass spectrometry to detect N-sulfo oversulfated chondroitin sulfate and to confirm the structure of the 2008 contaminant as oversulfated chondroitin sulfate and not oversulfated heparan sulfate.

Ultrasonic-assisted preparation of a low molecular weight heparin (LMWH) with anticoagulant activity

Low molecular weight heparin (LMWH) is currently used as an anticoagulant agent and constitutes an alternative to unfractionated heparin, which is the cause of serious adverse drug reaction such as heparin-induced thrombocytopenia (HIT). Commercially available LMWH is produced by enzymatic depolymerization that is costly or by chemical methods that are generally carried out under conditions that could imply side reactions that reduce final product efficiency and yields. In this work, we present the use of a physicochemical method for the production of LMWH. This method consists in the use of hydrogen peroxide-catalyzed radical hydrolysis assisted by ultrasonic waves. LMWH that are produced using this physicochemical method have an average molecular weight and anticoagulant properties (Anti-Xa and Anti-IIa) that are comparable to some of commercial LMWH that are currently used. Ultrasonic-assisted radical depolymerization of heparin leads to products with a remarkably low polydispersity index. Moreover, in comparison to other LMWH such as those produced by enzymatic β-elimination, this physicochemical depolymerization of heparin induces fewer oligosaccharides with less than five monosaccharide units. This contributes to the better preservation of the ATIII pentasaccharide binding sequence, which results in a high Anti-Xa/Anti-IIa ratio (1.86). However, LMWH obtained using this physicochemical method have a lower degree of sulfation than other LMWH, which seems to be the cause of a lower Anti-Xa and Anti-IIa activity (143.62±5.42 and 77.07±4.4, respectively).

On-line separation and characterization of hyaluronan oligosaccharides derived from radical depolymerization

Hydroxyl radicals are widely implicated in the oxidation of carbohydrates in biological and industrial processes and are often responsible for their structural modification resulting in functional damage. In this study, the radical depolymerization of the polysaccharide hyaluronan was studied in a reaction with hydroxyl radicals generated by Fenton Chemistry. A simple method for isolation and identification of the resulting non-sulfated oligosaccharide products of oxidative depolymerization was established. Hyaluronan oligosaccharides were analyzed using ion-pairing reversed phase high performance liquid chromatography coupled with tandem electrospray mass spectrometry. The sequence of saturated hyaluronan oligosaccharides having even- and odd-numbers of saccharide units, afforded through oxidative depolymerization, were identified. This study represents a simple, effective ‘fingerprinting’ protocol for detecting the damage done to hyaluronan by oxidative radicals. This study should help reveal the potential biological outcome of reactive-oxygen radical-mediated depolymerization of hyaluronan.

Human skin fibroblast response is differentially regulated by galactofucan and low molecular weight galactofucan

Abstract Wound healing is a sum of mechanisms that have to be finely modulated to reconstitute a tissue similar to the original. Several pathologies induce a decrease of healing rate and patients have to be treated to help the closure of wound. Glycosaminoglycan (GAG) mimetics have proven to be valuable in stimulating wound healing. In this study, galactofucan (fucoidan) was extracted from the brown seaweed Saccharina longicruris during two harvest periods (M05 and N05 fractions). Crude galactofucan M05 and N05 were subjected to radical depolymerization (RDP) for 4 h to obtain 2 RDP fractions (M05 RDP and N05 RDP fractions). Each fraction was added on cultured dermal fibroblasts and growth rate, collagen and MMP secretion were analyzed after treatment. Crude and RDP galactofucan influenced fibroblast growth, apoptosis and the synthesis of matrix metalloproteinase (MMP) and collagen according to the molecular weight of the polymer. Both crude galactofucan extracts reduced cell growth and increased MMP secretion, whereas only M05 stimulated apoptosis. By contrast, RDP fractions significantly increased collagen-I secretion and have no negative effect on fibroblast proliferation. Thus, crude and RDP galactofucan exhibit different metabolic activities on fibroblasts, which may be of interest in the area of wound healing.

Low molecular weight derivatives of different carrageenan types and their antiviral activity

A number of low molecular weight (LMW) fractions of carrageenans with different structural types were obtained by free radical depolymerization (H2O2), mild acid hydrolysis (HCl), and a specific enzyme. Three samples of carrageenans were depolymerized: kappa-carrageenan from Chondrus armatus, kappa-carrageenan from Kappaphycus alvarezii, and kappa/beta-carrageenan from Tichocarpus crinitus with initial molecular weights of 250, 390, and 400 kDa, respectively. The chemical depolymerization by two methods resulted to LMW derivatives of carrageenans with molecular weight from 1.2 to 3.5 kDa. Oligosaccharides of kappa- and kappa/beta-carrageenans with molecular weight of 2.2 and 4.3 kDa, respectively, were obtained after enzymatic depolymerization by recombinant kappa-carrageenase from Pseudoalteromonas carrageenovora. It was shown that the antiviral activity of high molecular weight carrageenans against tobacco mosaic virus was higher than that of their LWM derivatives independently on the depolymerization method. The method of depolymerization had some influence on the antiviral activity of carrageenan. LMW derivatives of kappa- and kappa/beta-carrageenans obtained by mild acid hydrolysis showed higher antiviral activity than the products of free radical depolymerization. The oligosaccharides prepared by enzymatic degradation possessed the lowest activity.

Rheological Behavior and Non-enzymatic Degradation of a Sulfated Galactan from Halymenia durvillei (Halymeniales, Rhodophyta)

The rheological behavior of a sulfated galactan extracted from Halymenia durvillei, a red seaweed collected in the coastal waters of a small island of Madagascar (Nosy-be in Indian Ocean), was investigated in dilute and semi-dilute solutions. In dilute solution with NaCl at 0.3M, the polysaccharide adopted a coil conformation whereas, at higher concentrations, the polymer had the behavior of shear-thinning fluid, typical of polymer with high molar mass or semi-rigid conformation. Degradations of this lambda carrageenan-like, using radical depolymerization, and high-pressure homogenization led to several samples of various and controlled molar masses. The measure of their intrinsic viscosities permitted the determination of the relationship of Mark-Houwink-Sakurada.

Low molecular fucoidan and its macromolecular complex with bee venom melittin

Low molecular weight (LMW) fucoidan, obtained by free radical depolymerization of high molecular polysaccharide extract of brown algae Hizikia fusiformis, was complexed with HPLC purified bee venom melittin. Water soluble form of the LMW fucoidan – melittin complex shows increased anti-inflammatory activity, inhibiting the production of nitric oxide in murine macrophage cell line Raw 264.7. The LMW fucoidan:melitin complex obtained in this study showed good biological activities, resulting in 2-fold reduction of the melittin toxicity. The fucoidan: melittin macromolecular complex obtained should be useful in future therapeutic applications.

Chain Degradation during Dissolution of Polymer-Fullerene Nanocomposites as a Result of Interaction of Entangled Polymer Matrix with the Filler

Chain degradation of poly(α-methylstyrene) and polystyrene during dissolution of their nanocomposites with fullerene C60 in solvents of different quality with respect to fullerene was studied in detail by size exclusion chromatography and UV spectroscopy. Chain ruptures have been shown to arise during swelling of composites but only for samples with entangled polymer matrix. The data obtained confirm that the hindered mobility of chains because of interaction of the entangled matrix with fullerene is the only cause of degradation. Chain rupture leads to radical depolymerization accompanied by covalent binding of fullerene with the chain fragments, which results in changing of the polymer matrix structure. Chain degradation indicates deterioration of the mechanical properties of the polymers in the presence of C60. The possibility of chain degradation in polymer-filler nanocomposites under deformation with the simultaneous observation of an apparent reinforcement effect because of the addition of filler in the polymer matrix is discussed.

Production of Arabinoxylan-oligosaccharides from Flaxseed (Linum usitatissimum)

Flaxseed mucilage from Linum usitatissimum L. species was constituted by arabinoxylan (about 75%) and pectin (about 25%). A new procedure was developed to obtain only arabinoxylans which implicated treatment of the pectin fraction by enzymatic hydrolysis with pectinase. Then three processes of depolymerization were evaluated on arabinoxylans. First, a thermic hydrolysis in mild acid conditions was performed and an ultrafiltration process was used as purification method. Second, the potential of xylanases from different glycoside hydrolase families for arabinoxylan-oligosaccharides (AXOS) production was tested, and finally a radical depolymerization was conducted. Average molecular weights were determined by high pressure size exclusion chromatography coupled with multiple angle laser light scattering (MALLS), and carbohydrate compositions were determined by high pH anion exchange chromatography pulse amperometric detector (HPAEC-PAD). Both chemical and enzymatic treatments were inefficient to convert arabinoxylans from flaxseed mucilage into AXOS. Only radical depolymerization process was allowed to obtain arabinoxylan-oligosaccharides presenting different molecular weights (11.9 x 10(3) to 1.9 x 10(3) g mol(-1)) with satisfactory yields (75% to 35%).

Preparation and characterization of molecular weight fractions of glycosaminoglycan from sea cucumber Thelenata ananas using free radical depolymerization

A glycosaminoglycan from sea cucumber Thelenata anana (THG) was isolated as a polymer of molecular weight of around 70 kDa. Its low molecular weight derivatives were first prepared by free radical depolymerization with hydrogen peroxide in the presence of copper(II) ion. The parameters of the process were investigated by a high-performance gel permeation chromatography. Analyses of chemical composition and molecular weight distribution indicated that the fragmentation of the main-chain of THG occurred randomly, obeyed pseudo first-order kinetics, and produced species with rather narrow and unimodal distribution of molar mass. The characterization of different molecular weight fractions was investigated by using viscometry and atomic force microscopy (AFM). Analysis of molecular weight and intrinsic viscosity in terms of the known theories for unperturbed wormlike cylinder yielded 1201 ± 110 nm −1, 15.3 ± 1.5 nm, and 1.5 ± 0.3 nm for molar mass per unit contour length M L, persistence length q, and diameter d, respectively. The M L and d values were approximately consistent with those observed by AFM. The present data suggest that THG may dissolve in 0.1 M aqueous NaCl as single-stranded helical chains.

Population-based models of thermoplastic degradation: Using optimization to determine model parameters

A comparison of different model forms for non-oxidative thermoplastic degradation rate is presented. A physically meaningful model is derived from a three-step radical depolymerization mechanism. A moment-based approximation to the population balance equations is derived and implemented to predict the thermal degradation of polyethylene (PE) and poly(methyl methacrylate) (PMMA). In the absence of accurate molecular dynamical calculations to predict the controlling model parameters, it is necessary to use experimental data to find the best fit parameter values. A sequential quadratic programming algorithm (SQP) was used to find the set of parameters that minimized the error between the model and sets of non-isothermal thermogravimetric (TG) data at different heating rates for PE and PMMA.

Identification and functional characterization of hyaluronic acid in degenerative joint diseases

Hyaluronic acid (HA), is a long polysaccharide chain that is made of repeating disaccharide units of N-acetylglucosamine and glucuronic acid. In osteoarthritis (OA) and rheumatoid arthritis, the molecular weight and concentration of HA in synovial fluid (SF) are reduced. This might result from abnormal biosynthesis by synovial type B cells and free radical depolymerization of the HA chain. The intra-articular treatment with HA is an accepted therapy for OA. However, the ideal candidate for intra-articular HA has yet to be defined and the exact mechanism of action of HA is unclear. Our study showed that the levels of ROS and acute phase protein profiles were reduced in patients under going HA treatment. HA could increase the cell viability of human chondrocytes under the high oxidative stress conditions also been determined. The primary receptor for HA is CD44, mediating both cell-cell and cell-matrix interactions. We postulate that the biological process initiated by HA-CD44 interaction results in changes in chondrocytes as well as in SF protein profiles. We used the proteomic approaches to investigate the biological functions of HA. There were 31 proteins, functioning in cell rescue, cell defense, cell stress, energy, anaerobic glycolysis, protein fate, cellular organization and cell cycle involved in this regulatory signaling. It suggests that HA involves in the antioxidative activity, anti-inflammation, anti-catabolic effect and cell growth regulation. This study will contribute to our understanding of HA functioning in the progressive degeneration of cartilage and to explore the clinical application of HA for degenerative joint diseases.

Preparation of a low-molecular weight fraction by free radical depolymerization of the sulfated galactan from Schizymenia binderi (Gigartinales, Rhodophyta) and its anticoagulant activity

Depolymerization of the sulfated galactan from Schizymenia binderi with H 2O 2 in the presence of copper (II) acetate afforded a homogeneous fraction (MW 8500) in 50% yield. Alkylation and NMR spectroscopy analysis of this fraction indicated the presence of an agaran–carrageenan hybrid polysaccharide composed of 3-linked β- d-galactopyranosyl residue partially sulfated at position O-2 and 4-linked α- d-galactopyranosyl and α- l-galactopyranosyl residues mainly sulfated at position O-3 and glycosylated at position O-2. Discreet anticoagulant activity, similar to that of the native polysaccharide was found in the derivative obtained by sulfation of this fraction.

Durability of perfluorinated ionomer membrane against hydrogen peroxide

The durability of perfluorinated ionomer membrane, Nafion ® 117, with various counter ions against hydrogen peroxide was investigated as a degradation factor of polymer electrolyte fuel cells (PEFC). After protonated Nafion membrane was tested in 30% H 2O 2 solution at 80 °C, small amounts of fluoride ion and sulfate ion, which are derived from the C F bonds and the sulfonic acid groups, respectively, of the membrane, were detected in the solution. This fact indicated a potential vulnerability of the electrolyte membrane to H 2O 2 formed in the cell. The durability of Nafion with alkali and alkaline earth metal ions as counter ions were similar to that of protonated Nafion, and hence these cations do not have any specific effects on membrane degradation. In contrast, the presence of ferrous and cupric ions as counter ions significantly enhanced the decomposition rate of Nafion. This is due to the formation of strongly nucleophilic radicals such as hydroxy and hydroperoxy radicals upon decomposition of H 2O 2 at these catalyst ions. The results of FT-IR and 19F NMR measurements of deteriorated Fe 2+-Nafion membrane revealed that both the main and side chains are decomposed at similar rates by radical attack, most probably because the decomposition proceeds through radical de-polymerization (so-called un-zippering mechanism).

Mechanisms Involved in Enhancement of Osteoclast Formation and Function by Low Molecular Weight Hyaluronic Acid

Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-kappa B ligand) in a mouse monocyte cell line (RAW 264.7). HA at M(r) < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at M(r) > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-kappa B) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.

On the theory of radical depolymerization: A rigorous solution

AbstractA rigorous solution to a model of radical depolymerization is presented. The model includes random chain scission, depropagation, radical transfer, and first‐order radical termination. The evolution of the molecular weight distribution in the course of depolymerization has been determined under the condition that the initial polymer is characterized by Flory's distribution. The kinetic equations consider the presence of chains with two radicalized ends, which are usually neglected. The commonly used simplifying assumption of the steady‐state radical concentration is not employed, and this makes the obtained results valid at any ratios between the rate constants. The predictions of the steady‐state approximation are compared to those of the rigorous approach in the case of depolymerization accompanied by volatilization of monomeric species. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 965–982, 2003