Preparation, physicochemical characterization and swelling properties of composite hydrogel microparticles based on gelatin and pectins with different structure.

Composite hydrogel microparticles based on pectins with different structures (callus culture pectin (SVC) and apple pectin (AU)) and gelatin were developed. Hydrogel microparticles were formed by the ionotropic gelation and electrostatic interaction of COO- groups of pectin and NH3+ groups of gelatin, which was confirmed by FTIR spectroscopy. The addition of gelatin to pectin-based gel formulations resulted in a decrease in gel strength, whereas increasing gelatin concentration enhanced this effect. The microparticle gel strength increased in proportion to the increase in the pectin concentration. The DSC and TGA analyzes showed that pectin-gelatin gels had the higher thermal stability than individual pectins. The gel strength, Ca2+ content and thermal stability of the microparticles based on gelatin and SVC pectin with a lower degree of methylesterification (DM) (14.8 %) were higher compared to that of microparticles based on gelatin and AU pectin with a higher DM (40 %). An increase in the SVC concentration, Ca2+ content and gel strength of SVC-gelatin microparticles led to a decrease in the swelling degree in simulated gastrointestinal fluids. The addition of 0.5 % gelatin to gels based on AU pectin resulted in increased stability of the microparticles in gastrointestinal fluids, while the microparticles from AU without gelatin were destroyed.

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.

Characterization and swelling properties of composite gel microparticles based on the pectin and κ-carrageenan.

The aim of this research was to produce composite gel microparticles based on the pectin (campion callus culture (SVC) and commercial apple (AU) pectins) and κ-carrageenan and investigate the relationship between the characteristics and swelling properties of the composite microparticles. The microparticles were obtained using emulsion dehydration techniques with successive incubation in calcium chloride solution. A significant positive correlation between the Ca2+ content and the SVC concentration in gel formulations was shown. Decreasing degree of methyl esterification (DM) of the SVC pectin promoted Ca2+ binding in comparison with the AU pectin. Increasing concentration of the pectin promoted increasing gel strength of the composite microparticles. The higher gel strength of the composite microparticles based on the SVC pectin was probably due to the lower DM and a higher linearity in comparison with the AU pectin. The microparticle gel formulations with a higher pectin concentration had a lower swelling degree in the simulated digestive fluids. The addition of the carrageenan to the gel formulations led to an increase in the swelling degree in comparison with that without carrageenan. The correlation analysis indicated that increasing initial Ca2+ content and gel strength of the microparticles promoted decreasing swelling degree of the composite microparticles.

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.

Preparation and release characteristics of mesalazine loaded calcium pectin-silica gel beads based on callus cultures pectins for colon-targeted drug delivery.

The aim of this work is to produce calcium pectin-silica gel beads containing mesalazine as a drug model in order to control the drug release in the colon. The mesalazine loaded calcium pectin-silica gel beads were prepared using the ionotropic gelation method. Energy-dispersive X-ray analysis revealed that increasing the Na2SiO3 concentration led to an increase of the silicon content on the surface and in the cross-sections of the beads. The addition of Na2SiO3 to the gel formulations made from the duckweed callus culture pectin led to a decrease in the swelling degree that appeared to be related to the higher gel strength of these beads. The beads made from pectins of campion and duckweed callus cultures with adding of 22.2 mg/ml of Na2SiO3 showed the lowest release of mesalazine in simulated gastric and intestinal fluids. An increase in the reaction time up to 60 min during incubation in the cross-linking solution of CaCl2 led to a slower release of drug from the beads. An elevated release of mesalazine was achieved in the simulated colonic fluid. Prepared calcium pectin-silica gel beads containing mesalazine as a drug model can be proposed for controlled drug release in the colon.

Adhesive properties of calcium pectinate gels prepared from callus cultures pectins.

The aim of this research is to investigate the influence of the surface morphology of the calcium pectinate gel (CaPG) beads as well as the physicochemical characteristics of pectins and the CaPG beads on the adhesive properties of gels against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Bacillus subtilis. The adhesion of the bacteria depends on the type of pectin and the surface morphology of the beads. The faster adhesion on CaPG beads appeared to be related to a lower degree of methyl esterification (DE), a higher molecular weight (Mw) and specific viscosity of the pectin and a higher gel strength. Surface roughness measurements were performed using an atomic force microscope. The beads from pectins with a higher Mw, a higher specific viscosity and a lower DE had a higher surface roughness. The surface roughness was one of the factors promoting adhesion of the bacteria onto the calcium pectinate gels. The surface morphology was observed under a scanning electron microscope (SEM). SEM images illustrated that E. coli and B. subtilis adhered on the beads with a rough surface. CaPG beads obtained from callus culture pectins can be proposed for the preparation of gels with adhesive and antiadhesive properties.

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.

Adjuvant effect of lemnan, pectic polysaccharide of callus culture of Lemna minor L. at oral administration.

A pectic polysaccharide, lemnan LMC, was extracted from the callus of duckweed Lemna minor L. and was tested for adjuvant properties at oral administration with protein antigen. Mice were orally immunized thrice with weekly interval with free hen's egg lysozyme or lysozyme with LMC. Lemnan LMC was shown to increase delayed type hypersensitivity and serum antilysozyme IgG responses. LMC was established to increase levels of both serum IgG1 and IgG2a subclasses. The concentration of malondialdehyde and myeloperoxidase activity were found to be higher in the tissue samples obtained from small intestine of mice immunized with mixture of lysozyme/LMC than those immunized with lysozyme only. Thus, lemnan appeared to be useful as the adjuvant for oral immunization.

Effect of calcium, phosphate and nitrogen on cell growth and biosynthesis of cell wall polysaccharides by Silene vulgaris cell culture.

Medium nutrients such as calcium, phosphorus, nitrogen and nitrate to ammonium ratio have significant influence on the growth, biosynthetic and biochemical characteristics of polysaccharides produced by Silene vulgaris (M.) G. cell culture. Cell growth and production of polysaccharides was limited by an absence of any of these components in the medium. Optimal growth of the callus and production of arabinogalactan were achieved at 1.5-4.5 microM calcium while the optimal production of pectin named silenan was observed at 3.0-4.5 microM. The phosphate contents in the medium in the range of 0.63-3.75 microM were favorable for callus growth. Production of silenan was maximal at 1.25-3.75 microM phosphate. Optimal growth of the callus was achieved at 30-90 microM nitrogen. Maximal production of silenan was observed at 60 microM nitrogen while the optimal production of arabinogalactan was at 90 microM nitrogen (at ratio of NH(4)(+):NO(3)(-) as 1:2). A presence both of nitrate and ammonium is needed for the silenan biosynthesis (the NH(4)(+):NO(3)(-) ratio as 1:1 and 1:2). Yields and volumetric production of arabinogalactan were maximal at deletion of ammonium from the nutrient medium (ratio 0:1). Absence of calcium or nitrogen in the medium leads to a decrease of the galacturonic acid residues in silenan. The galactose residues contents in arabinogalactan were decreased in the absence of nitrogen and calcium in the medium.

An alternate carbon source for enhancing production of polysaccharides by Silene vulgaris callus.

Pectin termed silenan and acidic arabinogalactan were isolated as cell-wall polysaccharides of Silene vulgaris callus in the presence of various carbon sources as components of the media. The maximum yields, productivity per litre of medium and production per day of acidic arabinogalactan, were achieved using glucose or galactose as the carbon source. Sucrose was found to increase the production of the polysaccharides. Yields, productivity and rate of production of arabinogalactan per day were decreased in the presence of arabinose. Yields of silenan, productivity and rate of production per day were closely related irrespective of the sugar used as the carbon source in the media (sucrose, glucose or galactose) and yields of silenan from the callus growing on arabinose were comparable. A concentration of sucrose in the 20-50 g/L range enhanced the biosynthesis of silenan and at 50 g/L the silenan contained the linear backbone and the ramified regions of the macromolecule.

Changes in cell wall polysaccharides of Silene vulgaris callus during culture.

In Silene vulgaris (M.) G. cell culture three growth phases were distinguished, namely, a lag phase, an exponential phase and a stationary phase. Pectin termed silenan and an acidic arabinogalactan were isolated as cell wall polysaccharides of S. vulgaris callus at the different growth phases during culture. Production of silenan as the galacturonan (or rhamnogalacturonan) core was observed at the beginning of the exponential phase and at the stationary phase of the callus growth. Arabinogalactan, containing the galacturonic acid residues, is formed at the exponential phase followed by attachment to the core of silenan in the middle of the exponential phase. The arabinogalactan constituent of silenan appeared to be destroyed gradually at the stationary growth phase. The monosaccharide compositions of silenan and arabinogalactan were determined at various phases of the callus growth. Silenan was found to be formed in maximum amounts at the exponential phase of the cell growth. Insignificant alterations of the yields of acidic arabinogalactan were found during culture while total productivity per litre of medium and rate of production per day of arabinogalactan were found to be maximal at the exponential phase of growth.