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.

Pectic polysaccharides of the fresh plum Prunus domestica L. isolated with a simulated gastric fluid and their anti-inflammatory and antioxidant activities.

A pectic polysaccharide, designated as PD, was extracted from fresh plums (Prunus domestica L.) with a simulated gastric fluid. Galacturonan, which was partially substituted with methyl and O-acetyl ester groups, and rhamnogalacturonan were the main constituents of the linear regions of the sugar chains of PD. The ramified region contained mainly 1,4-linked ß-d-galactopyranose residues and, to a lesser extent, 1,5-linked α-l-arabinofuranose residues. The separation of PD, by DEAE-cellulose column chromatography, yielded two pectic fractions: PD-1 and PD-2, eluted with 0.1 and 0.2 M NaCl, respectively. Enzymatic digestion of PD with 1,4-α-d-polygalacturonase yielded the fraction PD-E. The parent pectin PD and the PD-1 fraction were found to diminish the adhesion of peritoneal leukocytes at the concentrations of 0.05-1.0mg/ml. However, the PD-E fraction failed to have an effect on cell adhesion at the concentrations of 0.05-0.1mg/ml. PD, PD-1 and PD-E were found to inhibit the production of superoxide anion radicals by reducing xanthine oxidase activity by 38%, 97% and 47%, respectively. Therefore, the PD-1 fraction appeared to be an active fragment of pectic macromolecule isolated from fresh plum with a simulated gastric fluid.

Immunomodulatory activity of polysaccharides isolated from Clerodendrum splendens: beneficial effects in experimental autoimmune encephalomyelitis.

BACKGROUND: Extracts of leaves from Clerodendrum have been used for centuries to treat a variety of medicinal problems in tropical Africa. However, little is known about the high-molecular weight active components conferring therapeutic properties to these extracts. METHODS: Polysaccharides from the leaves of Clerodendrum splendens were extracted and fractionated by ion exchange and size-exclusion chromatography. Molecular weight determination, sugar analysis, degree of methyl esterification, and other chemical characterization of the fractions were performed. Immunomodulatory activity of the fractions was evaluated by determining their ability to induce monocyte/macrophage nitric oxide (NO), cytokine production, and mitogen-activated protein kinase (MAPK) phosphorylation. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice, and severity of EAE was monitored in mice treated with intraperitoneal (i.p.) injections of the most active polysaccharide fraction. Lymph nodes (LN) and spleen were harvested, and levels of cytokines in supernatants from LN cells and splenocytes challenged with myelin oligodendrocyte glycoprotein peptide were determined. RESULTS: Fractions containing type II arabinogalactan had potent immunomodulatory activity. Specifically, the high-molecular weight sub-fraction CSP-AU1 (average of 38.5 kDa) induced NO and cytokine [interleukin (IL)-1α, -1ß, -6, -10, tumor necrosis factor (TNF; designated previously as TNF-α), and granulocyte macrophage-colony stimulating factor (GM-CSF)] production by human peripheral blood mononuclear cells (PBMCs) and monocyte/macrophages. CSP-AU1-induced secretion of TNF was prevented by Toll-like receptor 4 (TLR4) antagonist LPS-RS, indicating a role for TLR4 signaling. Treatment with CSP-AU1 also induced phosphorylation of a number of MAPKs in human PBMC and activated AP-1/NF-κB. In vivo treatment of mice with CSP-AU1 and CSP-NU1 resulted in increased serum IL-6, IL-10, TNF, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α/CCL3, and MIP-1ß/CCL4. CSP-AU1 treatment of mice with EAE (50 mg/kg, i.p., daily, 13 days) resulted in significantly reduced disease severity in this experimental model of multiple sclerosis. Levels of IL-13, TNF, interferon (IFN)-γ, IL-17, and GM-CSF were also significantly decreased, whereas transforming growth factor (TGF)-ß was increased in LN cells from CSP-AU1-treated EAE mice. CONCLUSIONS: Polysaccharide CSP-AU1 is a potent natural innate immunomodulator with a broad spectrum of agonist activity in vitro and immunosupressive properties after chronic administration in vivo.

Structural studies of the pectic polysaccharide from Siberian fir (Abies sibirica Ledeb.).

The pectic polysaccharide named abienan AS-A was isolated from the wood greenery of Abies sibirica using dilute hydrochloric acid (pH 4.0) at 70°C. The structure of abienan AS-A was elucidated using sugar composition analysis, ion-exchange chromatography and partial acid hydrolysis followed by NMR spectroscopy. The linear region of abienan AS-A was shown to contain linear 1,4-α-D-galactopyranosyluronan partially substituted with methyl esters or 3-O-acetyl groups and rhamnogalacturonan blocks consisting of 1,4-α-D-galacturonan partially substituted with methyl ester groups and connected by 2-O-substituted α-rhamnopyranose residues. The branched region of abienan AS-A was found to be made of RG-I. The side chains of RG-I were shown to contain 1,4-ß-galactan and branched arabinan. Some 4-O-substituted ß-galactopyranose residues were shown to be attached to the 4-position of the 2-O-substituted α-rhamnopyranose residues of the RG-I backbone. The arabinan groups were made up of a 1,5-linked α-L-arabinofuranan backbone that was 3-O-, 2-O-, and 2,3-di-O-substituted with the terminal and 1,3-linked α-L-arabinofuranose residues.

Structural characterisation of the polysaccharides from endemic Mongolian desert plants and their effect on the intestinal absorption of ovalbumin.

Using successive extractions with water and 0.7% aqueous ammonium oxalate, pectic polysaccharides were isolated from the following plants growing in the arid climate of Mongolia (Gobi): saxaul Haloxylon ammodendron Maxim., rhubarb Rheum nanum Sievers, Nitraria sibirica Pall., Peganum harmala L. and almond Amygdalus mongolica Maxim. The data obtained exhibited the primary synthesis of the cell wall pectic polysaccharides but not the middle lamellae water-soluble pectins in plants growing in the dry climatic zone. Both α-(1→4)-D-galacturonan and α-(1→4)-D-galacturonan, which was substituted with methyl groups, were found to be backbone of pectins. The L-arabinofuranose residues were identified as the main components of ramified regions. The pectins from almond differed from other pectins due to a high arabinose content. The data from NMR spectroscopy and methylation analyses demonstrated that pectic polysaccharides from almond included terminal, (1→5)-, (1→3)-linked and 3,5-substituted L-arabinofuranose residues and a small terminal D-galactopyranose and 2,5- and 2,3,5-substituted L-arabinofuranose residue content. The pectic polysaccharides were found to decrease the absorption of ovalbumin (OVA) in the blood from the gut lumen. The serum OVA level was lower in mice fed with OVA mixed with the pectins compared with the control group, which was administered OVA alone.

Structure of pectic polysaccharides isolated from onion Allium cepa L. using a simulated gastric medium and their effect on intestinal absorption.

The polysaccharide fraction extracted with simulated gastric juice from onion bulbs contained a mixture of galactan with short-length sugar chains, pectic polysaccharides and evident content of proteinaceous material. Galacturonan and rhamnogalacturonan were the main constituents of the linear regions of the sugar chains of the pectic polysaccharides. The ramified regions included rhamnogalacturonan-I. NMR data revealed that the side chains of the ramified region contained mainly 1,4-linked ß-D-galactopyranose residues and lesser content of 1,3-linked ß-D-galactopyranose and 1,5-linked α-L-arabinofuranose residues. Furthermore, the proteinaceous material was determined to be partly linked to the sugar chains. The polysaccharide fraction was found to decrease absorption of ovalbumin (OVA) to the blood from the gut lumen. The serum OVA level was threefold lower in mice fed with OVA mixed with the onion pectins compared with the control group, which was administered OVA alone. Protein removal failed to abolish the inhibitory effect of the onion polysaccharides, confirming that the polysaccharide chains are the active component of onion gastric juice extract.

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.

Abrogation of the oral tolerance to ovalbumin in mice by citrus pectin.

OBJECTIVE: We studied the effects of dietary pectins (citrus pectin [CP] and apple pectin) on oral tolerance in mice. METHODS: Pectins (1 mg/d) were administered orally for 2 wk. Tolerance was induced with 20 mg of ovalbumin (OVA). Levels of serum antibodies (immunoglobulin [Ig] G, IgG1, IgG2a, IgE) and delayed type hypersensitivity response determined in footpad tests were measured after subcutaneous injection of OVA with complete Freund's adjuvant. Concentrations of immunoreactive OVA in blood were measured by enzyme-linked immunosorbent assay after feeding the animals 20 mg of OVA. Adhesion and cytokine production (tumor necrosis factor-alpha, interferon-gamma) were measured in peritoneal macrophages. RESULTS: Oral administration of CP was found to prevent the induction of immune hyporesponsiveness induced by OVA feeding. Animals fed OVA and CP were found to produce similar titers of antigen-specific serum IgG and levels of delayed type hypersensitivity response as those animals not fed OVA. CP increased levels of serum IgG1 and IgE. CP was found to enhance the penetration of immunogenic OVA into the serum. CP (1 mg/d) administered orally for 1 wk was also observed to enhance the adhesion and production of cytokines (tumor necrosis factor-alpha, interferon-gamma) in peritoneal macrophages. CONCLUSION: CP administered orally was shown to inhibit oral tolerance. Enhancement of protein antigen penetration to the blood and activation of macrophages were found to precede the inhibitory effect and appeared to mediate it.

Preventative antiinflammatory effect of potamogetonan, a pectin from the common pondweed Potamogeton natans L.

The pectic polysaccharide named potamogetonan (PN) was obtained using extraction of the leaves and stems of the common pondweed Potamogeton natans L. by an aqueous ammonium oxalate. The purified potamogetonan PN-300 was obtained using membrane ultrafiltration of PN and proved to be pectin with a molecular weight of 300 kDa. The capacity of potamogetonan PN-300 to prevent inflammation was assessed using a carrageenan paw edema test in mice. Oral administration of PN-300 24 h prior to induction of inflammation was found to reduce edema formation in a dose-related manner. The maximal effect of PN-300 was observed at 1 h after carrageenan injection (60% reduction of footpad swelling) and was comparable to that of indomethacin. The delayed edema (5 h) was less affected by pre-administration of PN-300 (33% reduction). PN-300 was found to improve the survival of mice subjected to a lethal dose of LPS. The anti-endotoxemic effect of PN-300 was shown to be mediated by decreased TNF-alpha and IL-1beta and increased IL-10 production.Thus, a pectin named potamogetonan PN-300 was isolated from P. natans and was shown to possess a preventive antiinflammatory effect following oral administration.

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.

Preventive effect of a pectic polysaccharide of the common cranberry Vaccinium oxycoccos L. on acetic acid-induced colitis in mice.

AIM: To study isolation and chemical characterization of pectin derived from the common cranberry Vaccinium oxycoccos L. (oxycoccusan OP) and the testing of its preventive effect on experimental colitis. METHODS: Mice were administrated orally with OP two days prior to a rectal injection of 5% acetic acid and examined for colonic damage 24 h later. Colonic inflammation was characterized by macroscopical injury and enhanced levels of myeloperoxidase activity measured spectrophotometrically with o-phenylene diamine as the substrate. The mucus contents of the colon were determined by the Alcian blue dye binding method. Vascular permeability was estimated using 4% Evans blue passage after i.p. injection of 0.05 mol/L acetic acid. RESULTS: In the mice treated with OP, colonic macroscopic scores (1.1+/-0.4 vs 2.7, P<0.01) and the total square area of damage (10+/-2 vs 21+/-7, P<0.01) were significantly reduced when compared with the vehicle-treated colitis group. OP was shown to decrease the tissue myeloperoxidase activity in colons (42+/-11 vs 112+/-40, P<0.01) and enhance the amount of mucus of colitis mice (0.9+/-0.1 vs 0.4+/-0.1, P<0.01). The level of colonic malondialdehyde was noted to decrease in OP-pretreated mice (3.6+/-0.7 vs 5.1+/-0.8, P<0.01). OP was found to decrease the inflammatory status of mice as was determined by reduction of vascular permeability (161+/-34 vs 241+/-21, P<0.01). Adhesion of peritoneal neutrophils and macrophages was also shown to decrease after administration of OP (141+/-50 vs 235+/-37, P<0.05). CONCLUSION: Thus, a preventive effect of pectin from the common cranberry, namely oxycoccusan OP, on acetic acid-induced colitis in mice was detected. A reduction of neutrophil infiltration and antioxidant action may be implicated in the protective effect of oxycoccusan.

Protective effect of comaruman, a pectin of cinquefoil Comarum palustre L., on acetic acid-induced colitis in mice.

The efficacy of comaruman CP, a pectin of marsh cinquefoil Comarum palustre L., was investigated using a model of acetic acid-induced colitis in mice. Mice were administered comaruman CP orally 2 days prior to rectal injection of 5% acetic acid and examined for colonic damage 24 hr later. Colonic inflammation was characterized by macroscopical injury, higher levels of myeloperoxidase activity, enhanced vascular permeability, and diminution of colonic mucus. Oral administration of comaruman CP was found to prevent progression of colitis. Colonic macroscopic scores and the total square of damage were significantly reduced in mice treated with CP compared with the vehicle-treated colitis group. Peroral pretreatment of mice with comaruman CP was shown to decrease tissue myeloperoxidase activity in colons compared with the colitis group. Comaruman CP was found to stimulate production of mucus by colons of normal and colitis mice. Comaruman CP decreased the inflammatory status of normal mice as elicited by reduction of vascular permeability and adhesion of peritoneal neutrophils and macrophages. Thus, a preventive effect of comaruman on acetic acid-induced colitis in mice was detected. Reduction of neutrophil infiltration and enhancement of colon-bound mucus may be implicated in the protective effect of comaruman.

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.

Characterisation of the oral adjuvant effect of lemnan, a pectic polysaccharide of Lemna minor L.

Lemnan LM, apiogalacturonanic pectin of duckweed Lemna minor L. was tested for adjuvant properties following oral administration with protein antigen. Male Swiss mice were orally immunized thrice with weekly intervals with free OVA or OVA with lemnan (LM). Lemnan LM was shown to increase delayed type hypersensitivity (DTH) and serum anti OVA IgG responses. LM was established to increase levels of both serum IgG1 and IgG2a subclasses, intestinal IgA and failed to elevate levels of serum IgE. Lemnan was found to increase the adhesion of macrophages and to enhance the generation of oxygen radicals by macrophages in response to phorbol 12-myristate 13-acetate. Serum OVA levels were four-fold higher in mice immunized with the mixture of OVA and LM in comparison with those in mice immunized with OVA only. Thus, substantial systemic and local mucosal immune responses were attained by oral immunization with the mixture of OVA and lemnan. Lemnan appeared to elicit adjuvant activity via induction of both Th1- and Th2-type responses. The immunopotentiating effect of lemnan may result from enhanced antigen ingestion and stimulation of macrophage activity.

Effect of lemnan, pectin from Lemna minor L., and its fragments on inflammatory reaction.

An effect of apiogalacturonanic pectin of duckweed Lemna minor L. (lemnan LM) was studied on the inflammatory response to ovalbumin injected intradermally into the footpad of control and ovalbumin-fed mice. Lemnan LM (1-2 mg per mouse) was found to enhance by as much as 50-60% the footpad swelling in control mice. Oral administration of ovalbumin was shown to result in sensitization that increased inflammation. Ovalbumin admixed with lemnan was found to increase by two-fold footpad edema in comparison with the mice receiving ovalbumin alone. Apple pectin used as a reference compound failed to influence the inflammatory reaction. Degradation of lemnan was performed to elucidate the active region of the polysaccharide macromolecule. The apiogalacturonanic fragment (LMP) obtained using a digestion of lemnan LM with pectinase was shown to increase the footpad response in both control and ovalbumin-fed mice. Fragment LMPH deprived of some terminal apiose residues as a result of partial acidic hydrolysis failed to have an effect on the inflammatory response.Thus, the data obtained reveal an enhancement by lemnan of the inflammatory response. The ramified apiogalacturonan seemed to be the active region of the lemnan macromolecule.

Forming and immunological properties of some lipopolysaccharide-chitosan complexes.

The complex formation of lipopolysaccharide (LPS) with chitosan (Ch) was demonstrated using sedimentation velocity analysis in the analytical ultracentrifuge, centrifugation in glycerol gradient and isopicnic centrifugation in cesium chloride. An addition of Ch to the Escherichia coli and Yersinia pseudotuberculosis LPS solutions was found to result in formation of the stable LPS-Ch complexes. The interaction is a complicated process and depends on time and reaction temperature, as well as on the molecular weight of chitosan. A stable LPS-Ch complex could be formed only after preliminary incubation of the initial components at an elevated temperature (37 degrees C). It should be noted that process of LPS complexation with Ch is accompanied by additional dissociating of LPS. The complex formation was shown to be a result not only of ionic binding, but also of other types of interactions. The interaction of Ch with LPS was shown to modulate significantly the biological activity of LPS. The LPS-Ch complex (1:5 w/w) was shown to possess much lower toxicity in a comparison with the parent LPS at injection to mice in the similar concentration. The LPS-Ch complex was shown to maintain an ability to induce of IL-8 and TNF, but induction of IL-8 and TNF biosynthesis by the LPS-Ch complex was lower than that by the parent LPS. The complex LPS-Ch, similarly to the parent LPS, was found stimulated the formation of the IL-8 in the dose-dependent manner in the human embryonal kidney cells (HEK 293 cells) transfected with TLR4 in combination with MD2.

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.

Structural studies of the pectic polysaccharide from duckweed Lemna minor L.

The pectic polysaccharide of duckweed Lemna minor L. termed lemnan (LM) was shown to contain the ramified, "hairy" region. Using partial acid hydrolysis and Smith degradation followed by NMR spectroscopy of the fragments obtained, some structural features of the hairy region of LM were elucidated. Partial acid hydrolysis of LM afforded the crude polysaccharide fraction LMH that was separated into two polysaccharide fractions: LMH-1 and LMH-2. In addition, the oligosaccharide fraction LMH-3 contained 97% D-apiose was obtained from the supernatant. A further more rigorous acidic hydrolysis of LMH led to the crude polysaccharide fraction LMHR which was separated in to two fractions: LMHR-1 and LMHR-2. Smith degradation of LMH afforded the polysaccharide fragment LMHS differed in low contents of apiose residues. Unfortunately, NMR-spectroscopy failed to provide significant evidence concerning the structure of LMH-1 due to the complexity of the macromolecule. The structure of the 1H/13C-NMR spectroscopy including the correlation 2D NMR spectroscopy. As a result, alpha-1,4-D-galactopyranosyluronan was confirmed to be the main constituent of the LM backbone. In addition, the ramified, "hairy" region of the macromolecule appeared to contain segments consisting of residues of terminal and beta-1,5-linked apiofuranose, terminal and alpha-1,5-linked arabinofuranose, terminal and beta-1,3- and beta-1,4- linked galactopyranose, the terminal and beta-1,4-linked xylopyranose, and beta-1,4-linked 2-mono-O-methyl xylopyranose. Analytical and NMR-spectral data of LMHS confirmed the presence of considerable amounts of the non-oxidized of 1,4-linked D-galactopyranosyl uronic acid residues. Thus, some side chains of the ramified region of lemnan appeared to attach to D-galactopyranosyl uronic acid residues of the backbone.

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.