Elena A. Günter

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.

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.

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.

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.

The production of class III plant peroxidases in transgenic callus cultures transformed with the rolB gene of Agrobacterium rhizogenes

The production of plant peroxidases by plant cell cultures is of great interest because of the potential for industrial applications. We used plant cell cultures overexpressing the rolB gene to produce increased amounts of plant class III peroxidases. The rolB gene ensured the stable and permanent activation of peroxidase activity in the transformed callus cultures of different plants. In particular, the total peroxidase activity in transformed Rubia cordifolia cells was increased 23-86-fold, and the abundance of the major peroxidase gene transcripts was increased 17-125-fold (depending on the level of rolB expression) compared with non-transformed control calli. The peroxidase-activating effect of rolB was greater than that of other peroxidase inducers, such as external stresses and methyl jasmonate.

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 hens 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.

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 β-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 pectins sugar composition, DE and Mw, while it appears to have an insignificant influence on intracellular and extracellular arabinogalactans.

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.

Action of β-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.