Effect of Callus Cell Immobilization on the Textural and Rheological Properties, Loading, and Releasing of Grape Seed Extract from Pectin Hydrogels.

The purpose of the present study was to prepare pectin hydrogels with immobilized Lemna minor callus cells and to identify the effect of cell immobilization on the textural, rheological, and swelling properties; loading; and releasing of grape seed extract (GSE) from the hydrogels. Hardness, adhesiveness, elasticity, the strength of linkage, and complex viscosity decreased with increasing cell content in the hydrogels based on pectin with a degree of methyl esterification (DM) of 5.7% (TVC) and during incubation in gastrointestinal fluids. An increase in the rheological properties and fragility of pectin/callus hydrogels based on pectin with a DM of 33.0% (CP) was observed at a cell content of 0.4 g/mL. TVC-based pectin/callus beads increased their swelling in gastrointestinal fluids as cell content increased. TVC-based beads released GSE very slowly into simulated gastric and intestinal fluids, indicating controlled release. The GSE release rate in colonic fluid decreased with increasing cell content, which was associated with the accumulation of GSE in cells. CP-based beads released GSE completely in the intestinal fluid due to weak textural characteristics and rapid degradation within 10 min. Pectin/callus hydrogels have the ability to preserve GSE for a long time and may have great potential for the development of proanthocyanidin delivery systems due to their novel beneficial physicochemical and textural properties.

Ca-Alginate Hydrogel with Immobilized Callus Cells as a New Delivery System of Grape Seed Extract.

The development of new delivery systems for polyphenols is necessary to maintain their antioxidant activity and targeted delivery. The purpose of this investigation was to obtain alginate hydrogels with immobilized callus cells, in order to study the interaction between the physicochemical properties of hydrogels, texture, swelling behaviour, and grape seed extract (GSE) release in vitro. The inclusion of duckweed (LMC) and campion (SVC) callus cells in hydrogels led to a decrease in their porosity, gel strength, adhesiveness, and thermal stability, and an increase in the encapsulation efficiency compared with alginate hydrogel. The incorporation of LMC cells (0.17 g/mL), which were smaller, resulted in the formation of a stronger gel. The Fourier transform infrared analyses indicated the entrapment of GSE in the alginate hydrogel. Alginate/callus hydrogels had reduced swelling and GSE release in the simulated intestinal (SIF) and colonic (SCF) fluids due to their less porous structure and the retention of GSE in cells. Alginate/callus hydrogels gradually released GSE in SIF and SCF. The faster GSE release in SIF and SCF was associated with reduced gel strength and increased swelling of the hydrogels. LMC-1.0Alginate hydrogels with lower swelling, higher initial gel strength, and thermal stability released GSE more slowly in SIF and SCF. The GSE release was dependent on the content of SVC cells in 1.0% alginate hydrogels. The data obtained show that the addition of callus cells to the hydrogel provides them with physicochemical and textural properties that are useful for the development of drug delivery systems in the colon.

Delivery system for grape seed extract based on biodegradable pectin-Zn-alginate gel particles.

Pectin-Zn-alginate gel particles from callus culture pectin with increased linearity and decreased rhamnogalacturonan I branching and degree of methylesterification had a higher gel strength and encapsulation capacity. An increase of the alginate concentration led to an increase in the particle gel strength. The grape seed extract (GSE) loaded and empty particles swelled slightly in the simulated gastric fluid (SGF) and gradually in the intestinal (SIF) fluid. The swelling degrees of the GSE-loaded and empty particles in the simulated colonic fluids (SCF) were decreased in the range SCF-7.0 (pH 7.0 + pectinase) > SCF-5.3 (pH 5.3 + pectinase) > SCF-2.3 (pH 2.3 + pectinase). The FTIR spectra indicated that GSE was embedded in the composite particles. Negligible leakage of GSE in SGF was shown. The increase in GSE release in SIF was due to the decrease in particle gel strength and increased swelling degree. The GSE release in fluids simulating the colon inflammation (SCF-2.3 and SCF-5.3) was similar, and it was lower than that in the SCF-7.0 simulating a healthy colon due to the increased gel strength. The percentage release of GSE increased slightly after exposure to different pH. Pectin-Zn-alginate hydrogel systems may be promising candidates for colon-targeted GSE delivery systems.

Physicochemical and swelling properties of composite gel microparticles based on alginate and callus cultures pectins with low and high degrees of methylesterification.

Composite gel microparticles based on alginate and callus culture pectins with low and high degrees of methylesterification or apple pectin were produced. By varying the chemical composition of the pectic samples and the ratio of alginate to pectin, the gel strength, morphology, and swelling properties of composite microparticles can be altered. The inclusion of increasing concentrations of alginate in gel formulations promoted an increase in the microparticle gel strength and the formation of a smoother surface microrelief independently of the pectin chemical composition. Microparticles based on the pectin with a low degree of methylesterification (DM) and a higher concentration of alginate exhibited an increased swelling degree in the simulated digestive fluids. Microparticles based on the pectin with high DM and low alginate concentration were destroyed in the simulated intestinal fluid within 1 h due to the low Ca2+ content, gel strength, and grooved and rough surface of these microparticles. An increase in alginate concentration of gel formulations based on pectin with high DM led to increased stability of the microparticles in the simulated intestinal and colonic fluids due to increased Ca2+ content, microparticle gel strength and degree of crosslinking.

Preparation and properties of the pectic gel microparticles based on the Zn2+, Fe3+ and Al3+ cross-linking cations.

The aim of this work was to evaluate the relationship between the cross-linking cation content in the microparticles, chemical characteristics of pectins and swelling properties of the gel microparticles based on the Zn2+, Fe3+ and Al3+ cross-linking cations. A significant negative correlation between the Zn2+ content and DM of pectin indicated that decreasing DM of pectin promoted Zn2+ binding. The microparticles from the pectins with a higher linearity had a higher content of Fe3+. The microparticles from the pectins with a lower Mw, branching of rhamnogalacturonan I, amount of RG, specific viscosity and a higher linearity had a higher content of Al3+. The content of the Fe3+ ions in the microparticles was higher than the Zn2+ content. The Al3+ content in the microparticles was lowest. The Fe3+ and Zn2+ ions, which are more electronegative, bind more strongly to pectin in comparison with the less electronegative Al3+ cations. The microparticles with a higher Zn2+, Fe3+, and Al3+ content had a lower swelling degree in the simulated digestive fluids. Moreover, the higher swelling of the microparticles can be due to their porous or wrinkled surface. Variation of specific cations and pectins can influence the functionality of the gel microparticles, in particular swelling properties.

Swelling behavior and satiating effect of the gel microparticles obtained from callus cultures pectins.

Gel microparticles were prepared from pectins of campion (SVCgel) and duckweed (LMCgel) callus cultures, as well as from commercial apple pectin (APgel) by emulsion dehydration techniques with successive ionotropic gelation. The morphology and swelling behavior of the microparticles were determined after successive incubation in simulated gastric (SGF), intestinal (SIF), and colonic (SCF) fluids. Both SVCgel and LMCgel microparticles were found to swell in SGF and SIF gradually, and at oral administration decreased food intake by laboratory mice during the first 5 h of free-feeding. The SVCgel microparticles demonstrated the higher stability in SCF within 24 h than LMCgel ones. Only the SVCgel microparticles were shown to decrease food intake by 24% during the 21 h of free-feeding and decreased body weight of mice by 4% during 24 h after oral administration. The APgel microparticles lost their shape in SIF, then fully disintegrated after 0.5 h of incubation in SCF, and failed to affect food intake or mice body weight. The data obtained indicated that sustainability and swelling of the gel microparticles from the SVC pectin in the colonic fluid may provide the stronger satiating effect compared to that of the LMCgel microparticles.

Encapsulated drug system based on the gels obtained from callus cultures modified pectins.

The aims of this research were to obtain modified pectins of callus cultures using various culture conditions, to evaluate the relationship between the chemical characteristics of pectins, the swelling behavior and the release of prednisolone from calcium pectinate gel (CaPG) beads. An increase of the calcium concentration in the culture media correlated significantly with the rhamnogalacturonan I (RG-I) branching of the pectin. The beads from the pectin with a higher RG-I branching had the lower prednisolone release in a gastric fluid. The beads produced from the pectins obtained from callus cultured with a higher calcium concentration showed the lower prednisolone release. The swelling of the CaPG beads from pectin with a lower molecular weight (Mw) or linearity occurred to a lower degree. All beads prepared from modified pectins showed a high stability and a slow liberation of prednisolone in the simulated gastric and intestinal fluids, whereas rapid drug release in a colonic fluid. An applied strategy involving modification of the pectic structure using the abiotic factors allows obtaining the pectic gels with modified functional properties, in particular, with enhanced gastric and small intestinal resistance and a low drug release. These CaPG beads can be applied as the carriers for colon delivery of the drugs.

Calcium pectinate gel beads obtained from callus cultures pectins as promising systems for colon-targeted drug delivery.

Low methyl-esterified pectins obtained from the cell walls of the campion (SV, SV>300), tansy (TV, TV>300) and duckweed (LM, LM>300) callus cultures and apple pectin (AP, Classic AU 701) were used as the carriers for colon delivery of prednisolone. The pectins with molecular weight more than 300kDa (SV>300, TV>300, LM>300) formed gels which exhibited the higher gel strength. The higher gel strength of these gels appeared to be related to the higher Mw and the lower degree of methylesterification (DE) of these pectins. Release aspects of prednisolone in the simulated gastric (pH 1.25), intestinal (pH 7.0) and colonic (pH 7.0+pectinase) media were investigated. The LM-5%, AP-3% and AP-5% beads destroyed in simulated intestinal medium probably due to the higher DE of the LM and AP pectins. The SV>300-3% and TV>300-3% prednisolone loaded bead systems showed a high stability at pH 1.25 and pH 7.0. Prednisolone release occurred in a larger extent in colonic medium due to the enzymatic erosion of the beads. The SV>300-3% and TV>300-3% particles showed a more controlled release that appeared to be related to the lower DE, rhamnogalacturonan content, rhamnogalacturonan I branching and the higher linearity and Mw of the TV>300 and SV>300 pectins, as well as to the higher gel strength. This in vitro study suggests that calcium pectinate gel beads obtained from callus cultures pectins can be proposed as potential systems for colon-targeted drug delivery.

Cell-wall polysaccharide composition and glycanase activity of Silene vulgaris callus transformed with rolB and rolC genes.

The aim of this research is to investigate the effects of the Agrobacterium rhizogenes rol genes on the composition of cell-wall polysaccharides and glycanase activity in the campion callus. The expression of the rolC gene reduces the yield of campion pectin, while the expression of the rolB or rolC gene inhibits the volumetric production of both pectin and intracellular arabinogalactan. The rol genes are involved in regulating the activity of glycanases and esterases, thereby contributing to the modification of polysaccharide structures, their molecular weight (Mw) and the degree of pectin methyl esterification (DE). The increase in pectin arabinose residue appears to be connected to a decrease in intracellular and extracellular α-l-arabinofuranosidase activity in transgenic campion calluses. In transgenic calluses expressing the rolB and rolC genes, the increase in pectin galactose residue is likely due to a decrease in ß-galactosidase activity. The decrease in the Mw of pectin and its d-galacturonic acid content appears to be connected to an increase in extracellular polygalacturonase activity. Finally, the increase in pectinesterase activity causes a decrease in the DE of pectin. Thus, the expression of rolB and rolC genes in campion callus has a considerable effect on pectin's sugar composition, DE and Mw, while it appears to have an insignificant influence on intracellular and extracellular arabinogalactans.

Swelling and morphology of calcium pectinate gel beads obtained from Silene vulgaris callus modified pectins.

The aim of this research is to investigate the swelling properties and morphology of the calcium pectinate gel (CaPG) beads made from pectins of campion callus cultured using various medium nutrients (carbon sources, concentration of sucrose, calcium and 2,4-dichlorophenoxyacetic acid (2,4-D)). Gelled spheres were prepared by ionotropic gelation. The mean diameter, total surface area and volume of the dried beads varied depending on the plant cell culture conditions. The swelling of dried CaPG beads in solutions with pH 2 and pH 4 was demonstrated to occur more slowly (within 4 or 24h) with increasing sucrose and calcium concentrations or in the absence of auxin. All beads swelled less when placed in acidic media (pH 2 and pH 4) and swelled most extensively in NaCl (pH 6). The surface morphology of the CaPG beads was demonstrated to depend on the presence of sugars, calcium and auxin in the plant cell culture medium used. The slow swelling of dried CaPG beads was apparently related to their grooved surfaces. An applied strategy involving changing the composition and concentration of media components altered the swelling behavior of the CaPG beads and enhanced the acid and water resistance of the resultant pectinate hydrogels in physiological environments. In particular, the swelling of Ca 4.5, 2,4-D0, Suc30 and Suc100 CaPG beads occurred more slowly.

Action of beta-galactosidase in medium on the Lemna minor (L.) callus polysaccharides.

The callus culture of duckweed cultivated on medium containing different concentrations of beta-galactosidase was shown to produce the following polysaccharides: pectin lemnan LMC, intracellular AG1, and extracellular AG2 arabinogalactans. The samples of lemnan with 46% galactose residue reduction and 9-46% increased galacturonic acid residue content were obtained at beta-galactosidase concentrations of 10(-3)-10(-1)mg/mL. The most substantial alterations in the sugar composition of pectin were found to occur in the fraction with a molecular mass of 100-300 kDa. Low concentrations of enzyme failed to influence the sugar composition of intracellular arabinogalactan, whereas high concentrations were shown to decrease the amount of arabinose residues in AG1 and to cause galactan formation. Extracellular galactan was found to be produced on the medium with 10(-1) and 1mg/mL beta-galactosidase whereas extracellular arabinogalactan AG2 was shown to be biosynthesized without beta-galactosidase or at a beta-galactosidase concentration of 10(-3)mg/mL. Alterations in the sugar composition of polysaccharides were shown to be connected with the increasing activity of alpha-l-arabinofuranosidase and beta-galactosidase, and with the decreasing activity of intracellular polygalacturonase.

Influence of ultraviolet-C on the compositions of cell-wall polysaccharides and carbohydrase activities of Silene vulgaris callus.

UV-C irradiation (254 nm) was found to enhance the secretion of some cell-wall-degrading enzymes, especially the following carbohydrases: beta-galactosidase, alpha-L-arabinofuranosidase, polygalacturonase, pectinesterase, cellulase, xylanase, and beta-xylosidase, in the campion callus, contributing thereby to an alteration in the polysaccharide structure. The relative amounts of the galactose and arabinose residues in pectin (silenan) and of arabinose in arabinogalactan of calli irradiated during the exponential phase were shown to decrease during the stationary phase. A decrease in the degree of SV methylesterification was found for the irradiated callus. These alterations were found to persist over a long period of culturing time. Decreasing the relative amounts of the arabinose residues in arabinogalactan and pectin and the galactose residues in silenan corresponded to increasing activity of alpha-L-arabinofuranosidase and beta-galactosidase, respectively, due to treatment with UV-C. UV-C irradiation may be used as a tool for modifying the structural features of the cell-wall polysaccharides, such as the relative amounts of galactose and arabinose residues in the side chains of polysaccharides, with the purpose of obtaining physiologically active polysaccharides with the desired properties and structural features.