Structure of KOH-soluble polysaccharides from сoniferous greens of Norway spruce (Picea abies): The pectin-xylan-AGPs complex. Part 1.

Using 7 % KOH, the polysaccharide PAK has been isolated from the coniferous greens of Norway spruce. PAK was found to contain predominantly arabinoglucuronoxylan, xyloglucan and arabinan, but also pectic polysaccharides, glucomannan and arabinogalactan proteins (AGPs), as determined by 1D/2D NMR analysis. It was found that fractionation of PAK on DEAE-cellulose resulted in simultaneous elution of pectins, arabinoglucuronoxylans and AGPs. It was evident that the content of 4-OMe-α-D-GlcpA and xylose, 1,4-ß-D-GlcpA, and T-ß-D-GlcpA increased with an increase in NaCl concentration. However, 1,4-α-D-GalpA content was almost independent of NaCl concentration, indicating unchanged pectic polysaccharide concentration. Interestingly, pectins extracted with 0.1-0.3 M NaCl solutions were richer in rhamnogalacturonan-I (RG-I) than those extracted with water and 0.01 M NaCl. Conclusion: The content of RG-I, AGPs and arabinoglucuronoxylan rises with rising NaCl concentration. An intense signal indicating an intermolecular linkage between the xylan and RG-I domains, i.e. that part of the arabinoglucuronoxylan is covalently bound to RG-I, is observed in the HMBC spectra of the polysaccharides obtained. The discovery here of a new relationship between rhamnogalacturonan I and xylan contradicts the prevailing cell wall model.

Structure of KOH-soluble polysaccharides from сoniferous greens of Norway spruce (Picea abies): The pectin-xylan-AGPs complex. Part 2.

Polymers containing arabinoglucuronoxylan, fucogalactoxyglucan, pectin and arabinogalactan proteins were obtained from PAK isolated from Norway spruce with 7 % KOH. The pectin core of PAK-I2-F-1 and PAK-I2-F-2 was dominated by RG-I, as treatment with 1,4-α-D-polygalacturonase resulted in almost complete removal of homogalacturonan. Interestingly, the above has not affected the co-fractionation of arabinoglucuronoxylan (AGX), arabinogalactan proteins and rhamnogalacturonan I (RG-I). Since pectin was mainly represented by RG-I, we concluded that xylan is specifically associated with RG-I. Correlations in the HMBC spectrum demonstrate intermolecular interactions between the α-L-Rhap (RG-I) and the Xyl (xylan), indicating a covalently bound AGX:RG-I complex via the Xyl-(1→4)-Rha bond: …→2)-[(2,4-ß-D-Xylp)-(1→4)]-[(α-D-GalpA-(1→2)]-α-L-Rhap-(1→4)-α-D-GalpA-(1→…. In PAK-H1-1-F-1 and PAK-H1-1-F-2, parts of RG-I and xylan were removed by enzymolysis. Part of the xylan was probably attached to the above-mentioned RG-I blocks. The removal of part of RG-I, xylan and the disappearance of the signal in the HMBC spectrum indicating the bond between RG-I and xylan confirms that part of the arabinoglucuronoxylan is covalently bound to RG-I. The observed glycosidic linkage contradicts the dominant PCW model in which pectin and hemicellulose polysaccharide networks are considered as independent components. It can be concluded that alkali-soluble xylan from Norway spruce was detected both in the free state and covalently bound to pectin.

Characterization of pectin-xylan-glucan-arabinogalactan proteins complex from Siberian fir Abies sibirica Ledeb.

Polysaccharide ASK was isolated from the Abies sibirica foliage by extraction with an aqueous KOH solution. ASK was shown to contain structurally different polymers such as arabinoglucuronoxylans, xyloglucans, glucomannans, arabinogalactan-proteins (AGPs). The pectic polysaccharides were also found in the alkaline extract of ASK and were represented by regions of homogalactorunan and rhamnogalactouronan-I whose side sugar chains were made up chiefly of highly branched 1,5-α-l-arabinan. The potential couplings between those polysaccharides were examined. Our studies showed simultaneous elution of pectin, xyloglucans, arabinoglucuronoxylans and AGPs, indicating that pectins can be covalently bound to the other cell-wall polysaccharides. NMR spectroscopy results revealed that the polysaccharides obtained by ion-exchange chromatography almost had no free reducing ends. These findings corroborate the conclusion that pectin, AGPs, glucan and xylan are bound together. The existence of the covalently bound complex of pectin-xylan-xyloglucan-AGP is suggested herein. Pectin and xylan are hypothesized to be covalently linked through RG-I regions.

Structural characteristics of oxalate-soluble polysaccharides from Norway spruce (Picea abies) foliage.

Structurally different polymers were derived from Picea abies foliage by successive extraction with water (PAW), HCl solution (PAA) and (NH4)2C2O4 solution (PAO). The P. abies foliage was found to contain basically low-methoxyl pectin extractable with an (NH4)2C2O4 solution. PAW was shown to comprise primarily arabinogalactan-proteins (AGPs); PAA was composed of mixed AGPs and pectic polysaccharides, with the latter prevailing; and polysaccharide PAO isolated in the highest yield included chiefly pectic polysaccharides. The major constituents of PAO were low-methoxyl and low-acetylated 1,4-α-d-galacturonan and partially acetylated RG-I. The sugar side chains of RG-I contained chiefly highly branched 1,5-α-l-arabinan and arabinogalactan type I as a minor constituent. RG-I whose side chains had 1,5-α-l-arabinan represented short regions alternating with non-acetylated and unmethylesterified galacturonan regions. In addition to pectins, polysaccharide PAO contained AGPs, xylanes and glucomannans, indicating that these polysaccharides are in an intimate interaction.

Structural studies of the pectic polysaccharide from fruits of Punica granatum.

The polysaccharide PGO containing 76 % of uronic acids, was obtained from peels and membranes of Punica granatum fruits by extraction to the aqueous solution of (NH4)2C2O4. The chemical structure of PGO was characterized by enzymatic and partial acid hydrolyses, Smith degradation and 1D/2D NMR spectroscopy. It has been found that PGO consisted mainly of highly methyl-esterified and lowly acetylated pectin. Backbone of the macromolecule was represented by 1,4-α-D-GalpA, 1,4-α-D-GalpA(OMe), 1,4-α-D-GalpA(OAc). The branched region PGO contained minor segments of partially acetylated rhamnogalacturonan-I (RG-I). RG-I side chains were comprised of highly branched 1,5-α-l-arabinan and segments of arabinogalactan type I. In addition to pectins, PGO contained the glucuronoxylans and xyloglucans, indicating a close interaction of these polysaccharides with each other in the cell wall. It was concluded that P. granatum fruit could be a promising source of pectic polysaccharides.

Structural studies of biologically active pectin-containing polysaccharides of pomegranate Punica granatum.

The polysaccharide PGW was isolated from peels and membranes of fruits of pomegranate P. granatum by hot water extraction. The methods of ion exchange chromatography, partial acid hydrolysis, enzymatic hydrolysis and NMR spectroscopy were employed to establish the major elements of its structure. It was shown that this polysaccharide contained polymers with different structures, the main components among which were pectic polysaccharides, represented mainly by highly methyl-esterified and lowly acetylated 1,4-α-d-galactopyranosyluronan and a minor amount of partially 2-O- and/or 3-O-acetylated RG-I. The side carbohydrate chains of the branched region of RG-I were mainly represented by terminal, 5-O-, 3-O-, 2,5-di-O-, 3,5-di-O- and 2,3,5-tri-O-substituted α-l-Araf residues indicating the presence of branched 1,5-α-l-arabinan and minor regions of 1,4-ß-d-galactan or arabinogalactan type I. The degree of methylation of the isolated pectins varied depending on the method of treatment. As a result, peels and membranes of pomegranate fruits can be recommended as a source of highly and lowly methyl-esterified pectic polysaccharides.

Structural characteristics of oxalate-soluble polysaccharides of Sosnowsky's hogweed (Heracleum sosnowskyi Manden).

Arabinogalactan proteins (AGP) and pectic polysaccharides were isolated from above-ground parts of Heracleum sosnowskyi. The structural study has shown that a linear region of the pectic macromolecules consists of 1,4-α-d-galactopyranosyluronan blocks partially methyl esterified and acetylated. The branched region consists of 3-O- and partially 2-O-acetylated rhamnogalacturonan I. Side chains of the RG-I backbone include the regions of arabinogalactan I and branched 1,5-α-l-arabinan. The carbohydrate part of AGP consists of arabinogalactan II with a 1,3-ß-d-Galp main chain. The side chains of the branched area of AG-II are composed of 1,6-ß-d-Galp, 1,5-, 1,3,5-α-l-Araf, 4-O-Me-ß-d-GlcA and 1,4-ß-d-GlcpA, and non-reducing ends residues of ß-d-Galp, α-l-Araf, α-l-Rhap and α-l-Fucp. The branch points of the main and side chains are formed by 3,6-di-O-substituted ß-d-Galp. It was found that at least a portion of pectin is probably covalently linked to AGP, wherein AGP is linked to RG-I, but not with galacturonan.

Structural characteristics of pectic polysaccharides and arabinogalactan proteins from Heracleum sosnowskyi Manden.

Polymers with different structures were isolated from the aboveground part of Heracleum sosnowskyi Manden. The sequential treatment of Heracleum with water, HCl solution, and (NH4)2C2O4 solution was observed to decrease the arabinogalactan proteins (AGP) content and increase the pectins content in the extracted polysaccharides. The linear region of the HSO-I polysaccharide having the highest yield was found to be composed mainly of partially methylesterified homogalacturonan fragments, whereas the branched region was made up of fragments of rhamnogalacturonan I whose core represents 1,2-α-L-rhamno-1,4-α-D-galacturonan. The carbohydrate side chains of the branched region are linked to the α-L-Rhap core residues via the 1,4-glycosidic bond and consist chiefly of T-ß-D-Galp, 1,4-ß-D-Galp and 1,6-ß-D-Galp residues indicating the presence of the 1,4-ß-D-galactan. NMR spectroscopy revealed the carbohydrate moiety of the AGP molecule to consist mainly of 1,3- and 1,3,6-ß-D-Galp residues. The side chains comprised 1,6-ß-D-Galp, terminal 4-O-Me-ß-D-GlcpA, and ß-D-Galp.

Extraction and structural characteristics of pectic polysaccharides from Abies sibirica L.

Structurally different pectins were isolated from the wood greenery of Abies sibirica L. by the sequential extraction with water (ASW), HCl solution (pH∼4) (ASA), and NH3 solution (pH∼8.5) (ASN). The GalA/Rha molar ratios for the ASW (15), ASA (8.9), and ASN (3.9) polysaccharides diminish in the order ASW>ASA>ASN, indicating a decrease in homogalacturonans and increase in rhamnogalacturonan I in this series. The ASWH, ASAH, and ASNH homogalacturonans derived by acid hydrolysis of ASW, ASA, and ASN have similar Mw 23.8, 21.1, and 18.9kDa, respectively, corresponding to a mean polymerization degree of 97-122 for the GalA residue. The macromolecule backbone of ASN was represented mainly by moieties of partially methylesterified homogalacturonan and partially 2-O- and/or 3-O-acetylated rhamnogalacturonan I. The carbohydrate side chains of the branched region are primarily made up of terminal, 1,5-O-, 1,3,5-di-O- and 1,2,3,5-tri-O-substituted α-L-Araf residues and terminal, 1,3-O- and 1,3,6-di-O-substituted ß-D-Galp residues. The currently known pectin models were refined.

Structural studies of arabinan-rich pectic polysaccharides from Abies sibirica L. Biological activity of pectins of A. sibirica.

Highly branched arabinan-rich pectic polysaccharides, containing 84% of arabinose, was extracted from wood greenery of Abies sibirica L. The structure of arabinan was studied by the 1D and 2D NMR spectroscopy. The macromolecule backbone was represented mainly by RG-I (molar ratio GalA:Rha ∼ 1.3:1) patterns with high degree of rhamnose branching. Side chains were comprised of 1,5-linked α-L-Araf residues (the major part of polymer mass), 1,3,5-di-O- and 1,2,3,5-tri-O-linked α-L-Araf residues, confirming the presence of highly branched 1,5-α-L-arabinan. Although most L-Araf were in α-anomeric form, minor terminal ß-L-Araf-(1 →... was detected. 1,4-ß-D-linked Galp residues found in the side chains account for minor AG-I or 1,4-galactan, as compared to arabinan. A tentative structure was proposed. Polysaccharides obtained from Siberian fir greenery were screened for biological activity. Galacturonan had a strongest stimulating effect on germination and growth rate of seeds, germs and roots of Triticum aestivum, Avena sativa, and Secale cereale.

Influence of abnormally high leptin levels during pregnancy on metabolic phenotypes in progeny mice.

Maternal obesity increases the risk of obesity in offspring, and obesity is accompanied by an increase in blood leptin levels. The "yellow" mutation at the mouse agouti locus (A(y)) increases blood leptin levels in C57BL preobese pregnant mice without affecting other metabolic characteristics. We investigated the influence of the A(y) mutation or leptin injection at the end of pregnancy in C57BL mice on metabolic phenotypes and the susceptibility to diet-induced obesity (DIO) in offspring. In both C57BL-A(y) and leptin-treated mice, the maternal effect was more pronounced in male offspring. Compared with males born to control mothers, males born to A(y) mothers displayed equal food intake (FI) but decreased body weight (BW) gain after weaning, equal glucose tolerance, and enhanced FI-to-BW ratios on the standard diet but the same FI and BW on the high-fat diet. Males born to A(y) mothers were less responsive to the anorectic effect of exogenous leptin and less resistant to fasting (were not hyperphagic and gained less weight during refeeding after food deprivation) compared with males born to control mothers. However, all progeny displayed equal hypothalamic expression of Agouti gene-related protein (AgRP), neuropeptide Y (NPY), and proopiomelanocortin (POMC) and equal plasma leptin and glucose levels after food deprivation. Leptin injections in C57BL mice on day 17 of pregnancy decreased BW in both male and female offspring but inhibited FI and DIO only in male offspring. Our results show that hyperleptinemia during pregnancy has sex-specific long-term effects on energy balance regulation in progeny and does not predispose offspring to developing obesity.

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.