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We studied the effects of peptides from Amaranthus cruentus L., Rauwolfia serpentina Benth., and citrus plants on contractile activity of the uterine myometrium in rats. Pectin substances inhibited spontaneous contractile activity of uterine muscles and abolished the stimulatory effect of oxytocin. Pectins had a synergistic effect with epinephrine and acetylcholine.
A blend matrix of crosslinked starch and pectin was prepared and characterized by infra-red (IR) spectroscopy, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). The prepared blends were investigated kinetically for water sorption studies and alpha-amylase induced degradation adopting a gravimetric procedure. Based on the experimental findings, a plausible mechanism including both diffusion and surface enhanced degradation was suggested and degradation profiles were interpreted. The influence of various factors such as chemical architecture of the blend, pH and temperature of alpha-amylase solution were examined for the swelling and degradation kinetics of crosslinked starch-pectin blends. The effect of concentration of enzyme solution was also studied on the degradation profile of the blends. A correlation was established between the extent of degradation and water imbibing capacity of the degrading blends.
High-performance size exclusion chromatography revealed that alkaline-extracted peach fruit pectin dissolved in 0.05 M NaNO3 comprised a hierarchy of at least four aggregated, macromolecular-sized species. Each of the three largest species was found to be comparable in length to the three smallest subunits of an interconnecting gel network visualized by transmission electron microscopy of pectin, rapidly dried from solution. The interconnecting subunits of the gel network were rods or segmented rods and the integrated network formed a circular gel about 1 micron in diameter. Shadowed samples prepared from 5 and 50 mM NaCl or aqueous glycerol solutions produced images of partially dissociated subunits.
Rats, mice, and hamsters were fed a fiber-free purified diet for 30 days and the activity of a number of cecal microbial enzymes was determined. Expressed per gram cecal content, azoreductase activity was greatest in preparations from the hamster and least from the mouse, and beta-glucosidase and beta-glucuronidase activities were least active from the rat. Nitroreductase was less active and nitrate reductase more active from the hamster in comparison to the other species. When expressed per kilogram body weight, bacterial activities were always greatest from the hamster. When the basal diet was supplemented with pectin (50 g/kg diet), nitrate reductase activity was increased six- to sevenfold per gram cecal content for rats and mice (tenfold when expressed per kilogram body weight), but there was no effect on the nitrate reductase activity of hamster microflora. Pectin also significantly increased beta-glucuronidase activity in rats, but significantly reduced the activities of the other enzymes in at least one of the three species.
Viscometric measurements were carried out on well-characterized apple, citrus, sugar-beet pectins in order to analyse the effect of the nature and the amount of substituents (methyl, amide, acetyl groups) and of the rhamnose content on the flexibility of the polymeric backbone. Through the dependence of the intrinsic viscosity with the ionic strength the flexibility parameter B was determined. B values between 0.072 and 0.017 indicate that pectins are relatively stiff molecules. However, an increase in flexibility is noticeable with the rise of the rhamnose content and of the amount of amide groups of the pectic acids. The flexibility is also sensitive to the degree of methylation.
In this paper, a cascade theory approach to biopolymer gelation is developed to describe variation of the shear modulus with temperature for thermoreversible gels. The broadness of this 'melting transition' is seen to depend crucially on the enthalpy of cross-linking, while the critical gel melting temperature is determined by additional factors such as the entropy of cross-linking, the polymer concentration, the molecular weight, and the number of cross-linking sites. On using the model to fit experimental data for specific gelatin and pectin systems, it was found that the behaviour of gelatin (sharp melting transition and low melting temperature) is a consequence of the large negative enthalpy and entropy of cross-linking, while, in contrast, the broad melting transition and high melting point of the pectin system are consistent with much smaller negative values for these parameters.
Pectin methylesterase (PME; EC 3.1.1.11) activities are widespread in bacteria, fungi, and plants. PME-mediated changes in cell wall pectin structure play important roles in plant development. Genome sequencing projects have revealed the existence of large PME multigene families in higher plants. Additional complexity for PME regulation arises from the presence of specific PME inhibitor proteins (PMEI) in plant cells. Several assay procedures for the determination of PME activity have been reported. However, previous protocols suffered from various limitations. Here we report a protocol for a coupled enzyme assay based on methanol oxidation via alcohol oxidase (AO; EC 1.1.3.13) and subsequent oxidation of formaldehyde by formaldehyde dehydrogenase (FDH; EC 1.2.1.3). This simple and robust assay allows the continuous monitoring of PME activity in the neutral pH range. Furthermore, as plant PMEIs do not interfer with AO and FDH activities, this assay is suitable for the characterization of the inhibition kinetics of PMEI.
Two polysaccharides, a pectin (Vk100A2b) and a pectic arabinogalactan (Vk100A2a) with mean Mw 2 x 10(4) and 1.15 x 10(6)Da, respectively, were isolated from the dried powdered roots of Vernonia kotschyana Sch. Bip. ex Walp. by hot water extraction followed by fractionation on DEAE-Sepharose fast flow and Sephacryl S-400 HR. The pectin showed low-complement fixation activity and no influence on proliferation of B or T cells, while the pectic arabinogalactan showed a potent, dose-dependent complement fixation activity and a T cell independent induction of B-cell proliferation. Both polysaccharides induced chemotaxis of human macrophages, T cells and NK cells. exo-alpha-L-arabinofuranosidase and exo-beta-D-galactosidase digestion followed by component sugar and methylation analysis indicated that Vk100A2a consisted of a highly branched rhamnogalacturonan core with approximately 50% of the rhamnose 1,2,4-substituted, side chains rich in terminal-, 1,5-linked and 1,3,5-branched arabinose and terminal-, 1,4-, 1,6-linked and 1,3,6-branched galactose. The enzyme resistant part of Vk100A2a still showed strong complement fixating activity, suggesting that this activity may at least in part be expressed by carbohydrate structures present in the enzyme resistant, inner portion of the polymer.
The deposition of alternating layers of pectin and chitosan at a solid surface was studied using surface plasmon resonance. The binding of biopolymer to the surface was irreversible over the time scales examined. The deposition was dependent on the flow rate through the measurement cell with mass transport limitation at lower flow rates. The thickness of the deposited layer was dependent on the biopolymer concentration and was particularly marked for pectin. This was consistent with a process of initial attachment, followed by a slower structural rearrangement, which was inhibited at high initial surface concentrations of adsorbed biopolymer. Sequential deposition resulted in the formation of multilayers with an essentially linear growth rate.
This work presents the optimized separation of pectin oligomers, their analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), their subsequent immobilization to supports, and our initial steps towards solid-support assisted sequencing. The ambient pressure strong anion-exchange resin Source 15Q combined with ammonium formate buffer (AF) was used for the separation of unsaturated and saturated pectic oligogalacturonides (OGAs) derived from enzymatic digestion of pectin. Routinely, multi-milligram quantities of defined sizes OGAs with DPs from 5 to 19 were produced in excellent purity (>95%). Elution of OGAs followed by direct analysis of the peak fractions by MALDI-TOF MS. Purified OGAs (DP 5-7) were chemoselectively immobilized onto aminooxy-terminated polyethylene glycol polyacrylamide (PEGA) supports. Solid-phase anchoring took place at the reducing end of the oligosaccharide and resulted in the formation of an oxime linkage. The very high coupling yields confirmed the general suitability of aminooxy-PEGA resins for the immobilization of OGAs of different lengths. The OGA-functionalized PEGA supports were subsequently treated with aq TFA at 40 or 60 degrees C, and the chemical degradation products released from the support were analyzed by ESIMS. In all cases, the original OGA was degraded into smaller oligomers of various sizes down to the monomer. This work illustrates some of the basic principles underlying a strategy ultimately aimed at solid-support assisted sequencing of oligosaccharides.
The purpose of this work was to develop hollow calcium pectinate beads for floating-pulsatile release of diclofenac sodium intended for chronopharmacotherapy. Floating pulsatile concept was applied to increase the gastric residence of the dosage form having lag phase followed by a burst release. To overcome limitations of various approaches for imparting buoyancy, hollow/porous beads were prepared by simple process of acid-base reaction during ionotropic crosslinking. The floating beads obtained were porous (34% porosity), hollow with bulk density<1 and had Ft50% of 14-24 h. In vivo studies by gamma scintigraphy determined on rabbits showed gastroretention of beads up to 5 h. The floating beads provided expected two-phase release pattern with initial lag time during floating in acidic medium followed by rapid pulse release in phosphate buffer. This approach suggested the use of hollow calcium pectinate microparticles as promising floating-pulsatile drug delivery system for site- and time-specific release of drugs acting as per chronotherapy of diseases.
Emulsion gel (EMG) beads of calcium pectinate capable of floating in the gastric condition were developed using an emulsion-gelation method and their release properties were investigated. Attempts to modify the drug release were made by applying some additives into the starting solution prior to bead formation, by hardening with glutaraldehyde, and by coating with polymer. The metronidazole-loaded EMG beads were found to float on simulated gastric fluid. Increasing the drug to pectin ratio in the beads slowed the drug release from the conventional and the EMG beads. However, the drug release from these beads was rapid, i.e., about 80% of drug loading released within 20-80 min. The additives (PEG10000, glyceryl monostearate and Eudragit L) had a slight, insignificant, effect on the drug release. Using 2% glutaraldehyde as a hardening agent prolonged the drug release. Coating the beads with Eudragit RL significantly sustained the drug release while the beads remained buoyant. The results suggest that EMG beads are suitable as a carrier for intragastric floating drug delivery and that their release behaviour could be modified by hardening with glutaraldehyde or by coating with Eudragit RL.
Gels formed in situ following oral administration of dilute aqueous solutions of pectin (1.0 and 1.5%, w/v) to rats were evaluated as vehicles for the sustained release of the expectorant drug ambroxol hydrochloride. The solutions contained calcium ions in complexed form, which on release in the acidic environment of the stomach caused gelation of the pectin. In vitro studies demonstrated diffusion-controlled release of ambroxol from the gels over a period of 6 h. A bioavailability of ambroxol of approximately 64% of that of a commercially available formulation could be achieved from gels containing an identical dose of ambroxol formed in situ in the stomachs of rats, with appreciably lower peak plasma levels and a sustained release of drug over a period of at least 6 h. The influence of added sorbitol (17%, w/v) on the rheological and drug release properties of the formulations has been examined.
A calcium-induced pectin gel bead (PB) containing pectin hydrolysate was prepared, and the drug release profiles and degradation properties of the PB were investigated in aqueous media. The stiff PB swelled in physiological saline and its drug release rate decreased with exposure to increasing concentrations of CaCl2 during preparation. And erosion of the PB was not observed in physiological saline. However, the PB did disintegrate in phosphate buffer (pH 6.8) and the rate of disintegration depended on the calcium chloride concentration used to prepare the PB. In addition, the drug release rate of the PB in buffer solution decreased as the rate of gel erosion declined. Consequently, it appears that the PB gel matrix is an effective medium by which to control the release of drug within the gastrointestinal tract.
Sediment samples were collected from different estuarine and marine areas along the West coast of India. Eighteen actinomycete cultures were isolated using starch casein agar and were screened for polygalacturonase activity by growing them on pectin-agar plates. Clear zones were visualized using 1% cetrimide. Out of the 18 strains screened ten cultures could effect hydrolysis of pectin. The above cultures were subjected to secondary screening under submerged fermentation. The actinomycete strain of Streptomyces lydicus was found to be a potent producer of polygalacturonase. Different growth media were screened for enzyme production and the best medium was selected for further studies. The crude enzyme was used for the treatment of raw banana fibers.
Arabinan and galactan side chains of sugar beet pectins are esterified by ferulic acid residues that can undergo in vivo oxidative reactions to form dehydrodiferulates. After acid and enzymatic degradation of sugar beet cell walls and fractionation of the solubilized products by hydrophobic interaction chromatography, three dehydrodiferulate-rich fractions were isolated. The structural identification of the different compounds present in these fractions was performed by electrospray-ion trap-mass spectrometry (before and after (18)O labeling) and high-performance anion-exchange chromatography. Several compounds contained solely Ara (terminal or alpha-1-->5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was assigned in some cases to the O-2 and in others to the O-5 of non-reducing Ara residues. One compound contained Gal (beta-1-->4-linked-dimer), Ara (alpha-1-->5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was unambiguously assigned to the O-2 of the non-reducing Ara residue and O-6 of the non-reducing Gal residue. These results provide direct evidence that pectic arabinans and galactans are covalently cross-linked (intra- or inter-molecularly) through dehydrodiferulates in sugar beet cell walls. Molecular modeling was used to compute and structurally characterize the low energy conformations of the isolated compounds. Interestingly, the conformations of the dehydrodiferulate-bridged arabinan and galactan fragments selected from an energetic criterion, evidenced very nice agreement with the experimental occurrence of the dehydrodiferulated pectins. The present work combines for the first time intensive mass spectrometry data and molecular modeling to give structural relevance of a molecular cohesion between rhamnogalacturonan fragments.
The mechanism of action of purified apple pectin methylesterase on pectin (degree of methoxylation: DM 75) and methoxylated homogalacturonans (DM 70 and 90) was studied at pH 7.0 (optimal pH of the enzyme) and at pH 4.5 (close to the pH of apple juice). Different interchain distributions of the free carboxyl groups were obtained at pH 7.0 and 4.5: high-performance ion exchange chromatography indicated a typical single chain mechanism at pH 7.0, but a mechanism differing from the single and multiple chain ones at pH 4.5. However, the same intrachain distribution of the newly demethoxylated galacturonic acid residues was observed for both pHs by 1H NMR. The high content of consecutive de-esterified or consecutive esterified galacturonic acid residues suggested that apple PME acted with a multiple attack mechanism on the pectic substrate. The degree of multiple attack of the enzyme was greater than or equal to 10-11.
Small-deformation oscillatory measurements were performed on pectin-sucrose-glucose syrup systems at a total level of solids of 81%, with the polysaccharide content being fixed at levels of industrial use (1%). The experimental temperature range was between 50 and - 50 degrees C. Analysis of the temperature dependence of viscoelastic processes by the equation of Williams, Landel, and Ferry provides values of fractional free volume for the temperatures covering the glass transition region. The shift factors used in the conversion of mechanical spectra into master curves were normalised at suitably different temperatures so that their temperature dependence becomes coincident. The treatment implies an iso-free-volume state and relates to changes in the monomeric friction coefficient with increasing levels of intermolecular interactions in the mixture. A free-volume related glass transition temperature was defined and manipulated markedly by introducing pectin of variable degrees of esterification to the sucrose-glucose syrup system.