Differences in pectic polysaccharides between carrot embryogenic and non-embryogenic calli

Summary Cell walls and media were obtained from three kinds of carrot cell culture, namely, embryogenic callus (EC), non-embryogenic callus (NC) and somatic embryos (SE), and analyzed for their sugar content and sugar composition by electrophoresis and gas chromatography. EC formed large cell clusters while NC formed small clusters. Observations under the light microscope revealed that the intercellular contacts in NC were much more limited than those in EC. The analysis of pectic polysaccharides revealed that the level of neutral sugars was higher than that of acidic sugars in EC, while the opposite was true in NC. Gaschromatographic analysis of neutral sugars in pectic fractions revealed that EC and SE were rich in arabinose, while NC was rich in galactose. On the basis of these results, we discuss the possible involvement of neutral sugars, and of arabinose and galactose in particular, in pectic polysaccharides in intercellular contacts.Abbreviations EC embryogenic callus - NC non-embryogenic callus - SE somatic embryo - MS Murashige and Skoog - PAS periodic acid-Schiff s reagent     

A xylogalacturonan whose level is dependent on the size of cell clusters is present in the pectin from cultured carrot cells

A substantial level of xylose was detected in the pectic polysaccharides that had been extracted from carrot (Daucus carota L.) calli and purified by gel-permeation and ion-exchange chromatography. The results of the removal of neutral sugar chains and -elimination indicated that the xylose was not included in the neutral sugar chains but was directly bound to a polygalac-turonic-acid backbone. Methylation analysis confirmed that the xylose was directly linked to galacturonic acid at position 2 or 3, as a terminal residue. The amount of xylose was positively correlated with the size of cell clusters in several lines of cultured carrot cells.Abbreviations EC embryogenic callus - 4-GalA 4-linked galacturonic acid - NC non-embryogenic callus - T-Xyl terminal xylose - 3,4-GalA 3,4-linked galacturonic acid - 2,4-GalA 2,4-linked galacturonic acid Part of this work was supported by a research grant from the Science and Technology Agency of Japan and a Grand-in-Aid from the Ministry of Education, Science and Culture, Japan. The authors are grateful to Dr. Koichi Kakegawa of the Forestry and Forest Products Research Institute for his encouragement throughout this research.     

High molecular weight constituents from roots of Echinacea pallida: an arabinogalactan-protein and an arabinan

This investigation shows structural features of two macromolecules from roots of Echinacea pallida (Nutt.) Nutt: an arabinogalactan-protein (AGP) and an arabinan. The arabinogalactan-protein was precipitated with beta-glucosyl Yariv reagent from a high molecular weight fraction. Investigations of the neutral sugar composition revealed Gal (52.1% w/w) and Ara (38.2% w/w) in a ratio of 1.4:1, accompanied by Glc (6.9% w/w) and Rha (2.8% w/w). The content of uronic acids was 6.2%. Mild acid hydrolysis detects Ara and Glc being located at the periphery of the molecule. Linkage analyses and NMR spectroscopy revealed a backbone of the polysaccharide mainly consisting of 3-linked and 3,6-linked Galp-residues. Side chains are composed of 3,6-linked or 6-linked Galp terminating in 5-linked Araf, terminal Araf, Glcp and GlcAp. The protein part (3.9% w/w) of the AGP is rich in Hyp, Ser, Ala, Thr, Glu, Asp and Gly. The amount of Hyp was determined by a colorimetric method and found to be (0.65% (w/w) of the AGP, which is in good agreement with the result obtained by amino acid hydrolysis (0.67% w/w). The arabinan was isolated from the supernatant of the Yariv precipitation on the basis of solubility in EtOH (80%). It mainly consists of Ara (85.8%). Linkage analyses and NMR spectroscopy indicate a highly branched molecule, consisting of 3,5-linked, 5-linked and terminal Araf-residues in equal amounts.     

Isolation from sugar beet cell walls of arabinan oligosaccharides esterified by two ferulic acid monomers

Side chains of sugar beet (Beta vulgaris) pectins, which are mainly composed of arabinose (Ara) and galactose (Gal) residues, are esterified by ferulic acid units. Enzymatic hydrolysis of beet cell walls yielded several feruloylated oligosaccharides, which were separated by hydrophobic interaction chromatography. Two new oligomers were isolated in the fraction eluted by 25:75 (v/v) ethanol:water. An arabinotriose and an arabinotetraose esterified by two ferulic acid residues were obtained, and their structure was elucidated by mass spectrometry. It is shown that feruloyl groups are linked to O-5 of Ara residues, in addition to the known O-2 position. This work establishes for the first time, to our knowledge, that two neighboring Ara units may be esterified by two ferulic acid units. This close proximity may have important biochemical implications.     

The isolation, preliminary structural studies, and physiological activity of water-soluble polysaccharides from the squeezed berries of the snowball treeViburnum opulus

Water-soluble polysaccharide fractions VO1–VO4 were isolated from the squeezed berries of the snowball tree (Viburnum opulus) by successive extraction with water at various temperatures and pH and with aqueous solutions of ammonium oxalate. These fractions were purified by ion-exchange chromatography on DEAE cellulose, and the homogeneity of the purified polysaccharides was determined by gel filtration on Sephacryl S-500. Acidic polysaccharides close to pectins in their sugar composition were found in all the extracts (fractions VO1-1, VO2-1, VO3-2, and VO4-2). Residues of galacturonic acid, galactose, arabinose, and (to a lesser extent) rhamnose are their main constituents. Neutral polysaccharides composed mainly of galactose and mannose residues were additionally found in fractions extracted with acidified water (pH 4.0) and with aqueous ammonium oxalate solutions. Partial acidic hydrolysis and digestion with pectinase of acidic polysaccharides indicated that their carbohydrate backbone consists of α-1,4-linked residues ofD-galacturonic acid. NMR spectra of acidic polysaccharides (fractions VO3-2 and VO3-3) confirmed this and demonstrated that their side oligosaccharide chains are composed of β-1,4-linked galactopyranose residues and of terminal and 2,5- and 3,5-substituted residues of α-arabinofuranose at a Gal : Ara ratio of 3 : 1. Some polysaccharides fromV. opulus were found to possess an immunostimulating activity: they enhance phagocytosis, in particular, the phagocytic index and the secretion of lysosomal enzymes with peritoneal macrophages. Calcium ions were found to be necessary for the appearance of the stimulating effect of acidic polysaccharides fromV. opulus.     

Water-soluble polysaccharides from Ginkgo biloba leaves.

The water-soluble polysaccharides from dried Ginkgo biloba leaves were isolated after exhaustive extraction with organic solvents. The polysaccharide mixture could be separated into a neutral (GF1) and two acidic (GF2 and GF3) polysaccharide fractions by ion exchange chromatography. According to the Mr distribution GF1 and GF3 seemed to be homogenous, whereas GF2 could be further fractionated into two subfractions (GF2a and GF2b) by gel permeation chromatography. GF1 (Mr 23,000) showed the structural features of a branched arabinan. The main chain was composed of 1,5-linked arabinose residues and three in 12 arabinose molecules were branched via C-2 or C-3. GF2a (Mr 500,000) consisted mainly of 1,2,4-branched mannose (29%), 1,4-linked glucuronic (32%) and galacturonic (8%) acid as well as terminal rhamnose (25%). After removal of ca 70% of the terminal rhamnose the remaining polysaccharide showed a decrease in 1,2,4-branched mannose and an increase in 1,2-linked mannose indicating that at least half of the rhamnose residues were linked to mannose via C-4. GF3 (Mr 40,000) consisted of 1,4-linked galacturonic (30%) and glucuronic (16) acid, 1,3,6-branched galactose (15%), 1,2-linked (5%) and 1,2,4-branched (3.5%) rhamnose as well as 1,5-linked arabinose (11%). Rhamnose (5%) and arabinose (10%) were present as terminal groups. Mild acid hydrolysis selectively cleaved arabinose and the remaining polysaccharide showed an increased amount of 1,6-linked and terminal galactose and a decreased quantity of 1,3,6-branched galactose. These results indicated that the terminal as well as the 1,5-linked arabinose were mainly connected to galactose via C-3. The GF3 polysaccharide appeared to be a rhamnogalacturonan with arabinogalactan side chains.     

Sugar beet (Beta vulgaris) pectins are covalently cross-linked through diferulic bridges in the cell wall

Arabinan and galactan side chains of sugar beet pectins are esterified by ferulic acid residues that can undergo in vivo oxidative reactions to form dehydrodiferulates. After acid and enzymatic degradation of sugar beet cell walls and fractionation of the solubilized products by hydrophobic interaction chromatography, three dehydrodiferulate-rich fractions were isolated. The structural identification of the different compounds present in these fractions was performed by electrospray-ion trap-mass spectrometry (before and after (18)O labeling) and high-performance anion-exchange chromatography. Several compounds contained solely Ara (terminal or alpha-1-->5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was assigned in some cases to the O-2 and in others to the O-5 of non-reducing Ara residues. One compound contained Gal (beta-1-->4-linked-dimer), Ara (alpha-1-->5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was unambiguously assigned to the O-2 of the non-reducing Ara residue and O-6 of the non-reducing Gal residue. These results provide direct evidence that pectic arabinans and galactans are covalently cross-linked (intra- or inter-molecularly) through dehydrodiferulates in sugar beet cell walls. Molecular modeling was used to compute and structurally characterize the low energy conformations of the isolated compounds. Interestingly, the conformations of the dehydrodiferulate-bridged arabinan and galactan fragments selected from an energetic criterion, evidenced very nice agreement with the experimental occurrence of the dehydrodiferulated pectins. The present work combines for the first time intensive mass spectrometry data and molecular modeling to give structural relevance of a molecular cohesion between rhamnogalacturonan fragments.     

The isolation, preliminary study of structure and physiological activity of water-soluble polysaccharides from squeezed berries of Snowball tree Viburnum opulus

Water-soluble polysaccharide fractions VO1-VO4 were isolated from the squeezed berries of snowball tree (Viburnum opulus) by successive extraction with water at various temperatures and pH and with aqueous solutions of ammonium oxalate. These fractions were purified by ion-exchange chromatography on DEAE cellulose, and the homogeneity of the purified polysaccharides was determined by gel filtration on Sephacryl S-500. Acidic polysaccharides close to pectins in their sugar composition were found in all the extracts (fractions VO1-1, VO2-1, VO3-2, and VO4-2). Residues of galacturonic acid, galactose, arabinose, and (to a lesser extent) rhamnose are their main constituents. Neutral polysaccharides composed mainly of galactose and mannose residues were additionally found in fractions extracted with acidified water (pH 4.0) and with aqueous ammonium oxalate solutions. Partial acidic hydrolysis and digestion with pectinase of acidic polysaccharides indicated that their carbohydrate backbone consists of alpha-1,4-linked residues of D-galacturonic acid. NMR spectra of acidic polysaccharides (fractions VO3-2 and VO3-3) confirmed this and demonstrated that their side oligosaccharide chains are composed of beta-1,4-linked galactopyranose residues and of terminal and 2,5- and 3,5-substituted residues of alpha-arabinofuranose at a Gal: Ara ratio of 3:1. Some polysaccharides from V. opulus were found to possess an immunostimulating activity: they enhance phagocytosis, in particular, the phagocytic index and the secretion of lysosomal enzymes with peritoneal macrophages. Calcium ions were found to be necessary for the appearance of the stimulating effect of acidic polysaccharides from V. opulus.     

Structural characterisation of an anti-complementary arabinogalactan from the roots of Angelica acutiloba Kitagawa

An anti-complementary arabinogalactan (AGIIb-1), isolated from the roots of Angelica acutiloba Kitagawa, has been subjected to methylation analysis, digestion with alpha-L-arabinofuranosidase, controlled Smith-degradation, and partial acid hydrolysis. AGIIb-1 consisted of arabinose, galactose, rhamnose, galacturonic acid, and glucuronic acid in the molar ratios 1.8-2.2:1.0:0.2-0.3:0.2-0.4:0.1. AGIIb-1 contained mainly an arabino-3,6-galactan moiety, and most of the Ara was present as alpha-L-arabinofuranosyl residues in the non-reducing terminals and the highly polymerised and branched side-chains which were attached mainly to positions 3 and 6 of (1----6)- and (1----3)-linked Gal, respectively. Some Ara-containing chains were also attached to (1----4)-linked Gal residues. The 13C-n.m.r. data for AGIIb-1 showed that the Galp was beta. Mild acid hydrolysis of AGIIb-1 yielded several linear and highly branched arabino-oligosaccharides, a neutral arabinogalactan, and two acidic arabinogalactans. Some arabino-oligosaccharides contained a (1----4)-linked Arap at the reducing terminal. The neutral arabinogalactan contained (1----3)-, (1----4)-, and (1----6)-linked and 3,6-di-O-substituted Gal, whereas the acidic arabinogalactans contained, in addition, non-reducing terminal GlcA, (1----4)-linked GalA, and 2,4-di-O-substituted Rha. The anti-complementary activity was decreased when AGIIb-1 was partially hydrolysed with mild acid (10mM HCl, 100 degrees, 10 min), but treatment with exo-alpha-L-arabinofuranosidase markedly enhanced the activity.     

Substrate specificity of the alpha-L-arabinofuranosidase from Rhizomucor pusillus HHT-1

The alpha-L-arabinofuranosidase (AF) from the fungus Rhizomucor pusillus HHT-1 released arabinose at appreciable rates from (1-->5)-alpha-L-arabinofuranooligosaccharides, sugar beet arabinan and debranched arabinan. This enzyme preferentially hydrolyzed the terminal arabinofuranosyl residue [alpha-(1-->5)-linked] of the arabinan backbone rather than the arabinosyl side chain [alpha-(1-->3)-linked residues]. The enzyme-hydrolyzed arabinan reacted at and debranched the arabinan almost at the same rate, and the degree of conversion for both cases was 65%. Methylation analysis of arabinan showed that the arabinosyl-linkage proportions were 2:2:2:1, respectively, for (1-->5)-Araf, T-Araf, (1-->3, 5)-Araf and (1-->3)-Araf, while the ratios for the AF-digested arabinan shifted to 3:1:2:1. Enzyme digestion resulted in an increase in the proportion of (1-->5)-linked arabinose and a decrease in the proportion of terminal arabinose indicated this AF cleaved the terminal arabinosyl residue of the arabinan back bone [alpha-(1-->5)-linked residues]. Peak assignments in the 13C NMR spectra also confirmed this linkage composition of four kinds of arabinose residues. Both 1H and 13C NMR spectra are dominated by signals of the alpha-anomeric configuration of the arabinofuranosyl moieties. No signals were recorded for arabinopyranosyl moieties in the NMR spectra. Methylation and NMR analysis of native and AF-digested arabinan revealed that this alpha-L-arabinofuranosidase can only hydrolyse alpha-L-arabinofuranosyl residues of arabinan.     

Absence of arabinan in the side chains of the pectic polysaccharides strongly associated with cell walls of Nicotiana plumbaginifolia non-organogenic callus with loosely attached constituent cells

When leaf disks from haploid plants of Nicotiana plumbaginifolia Viv. were transformed with T-DNA and cultured on shoot-inducing medium, nonorganogenic callus. designated nolac (for non-organogenic callus with loosely attached cells), appeared on approximately 7% of leaf disks. In contrast, normal callus was generated on T-DNA-transformed leaf disks from diploid plants and on non-transformed leaf disks from haploid and diploid plants. Transmission electron microscopy revealed that the middle lamellae and the cell walls of one line of mutant callus (nolac-H14) were barely stained by ruthenium red. even after demethylesterification with NaOH, whereas the entire cell wall and the middle lamella were strongly stained in normal callus. In cultures of nolac-H14 callus, the level of sugar components of pectic polysaccharides in the hemicellulose fraction was reduced and that in the culture medium was elevated, as compared with cultures of normal callus. These results indicate that pectic polysaccharides are not retained in the cell walls and middle lamellae of nolac-H14 callus. In nolac-H14, the ratio of arabinose to galactose was low in the pectic polysaccharides purified from all cell wall fractions and from the medium, in particular, in the hemicellulose fractions. The low levels of arabinofuranosyl (T-Araf, 5-Araf, 2,5-Araf, and 3,5-Araf) residues in the pectic polysaccharides of the hemicellulosic fraction of nolac-H,14 indicated that no neutral-sugar side chains, composed mainly of linear arabinan. were present in nolac-H14. Arabinose-rich pectins. which are strongly associated with cellulose-hemicellulose complexes, might play an important role in intercellular attachment in the architecture of the cell wall.     

Studies on the structure of a lithium-treated soybean pectin: characteristics of the fragments and determination of the carbohydrate substituents of galacturonic acid

Two galacturonic-acid-containing polysaccharide fractions (ChSS and P) were isolated from soybean meal and subjected to lithium treatment. The fragments obtained were analyzed by using monosaccharide and methylation analyses, and NMR spectroscopy. Lithium degradation of ChSS, followed by sodium borodeuteride reduction, hydrolysis, sodium borohydride reduction, and acetylation afforded alditol acetates, of which the labeled ones reflected residues linked to GalA. As followed from quantifications of the labeled and non-labeled alditols from each constituent monosaccharide by GLC-EIMS, 6 mol% of Ara, 22 mol% of Fuc, 13 mol% of Gal, 53 mol% of Rha, and 57 mol% of Xyl are glycosidically linked to GalA. Analysis of the lithium-treated polymer revealed that it contains arabinogalactan side chains linked to Rha O-4, which consist of a beta-(1 --> 4)-linked galactan substituted with highly branched arabinan chains. On average, an arabinogalactan chain contains up to 29 Gal and 25 Ara residues. Surface plasmon resonance was used to determine conditions for affinity chromatography. Furthermore, this technique confirmed the presence of terminal alpha-Fuc residues in ChSS. Polysaccharide P turned out to be relatively resistant to lithium degradation.     

Water-soluble polysaccharide from Bupleurum chinense DC: Isolation, structural features and antioxidant activity

The water-soluble polysaccharide (BCPS-1) was isolated from Bupleurum chinense DC. BCPS-1 (M_w = 29 kDa) was composed of Ara; Gal; Glc with a molar ratio of 2.1:2.5:1. According to FT-IR, partial acid hydrolysis, periodate oxidation and Smith degradation, methylation and GC-MS analysis, the results indicate BCPS-1 had a backbone of (1→5)-linked Ara, (1→4)-linked Gal and (1→3)-linked Gal residues with occasionally branches at O-6. The branches were composed of (1→4)-linked Glc, and terminated with Gal residues. The in vitro antioxidant activity evaluated by DPPH radical scavenging method showed that BCPS-1 had a significant antioxidant effect in a concentration-dependent manner.     

High levels of non-methylesterified pectins and low levels of peripherally located pectins in loosely attached non-embryogenic callus of carrot

Carrot embryogenic callus (EC) forms larger and tighter clusters of cells than does non-embryogenic callus (NC). Morphological and histochemical analyses of EC and NC were made using the electron microscope. The entire cell wall in NC was strongly stained by ruthenium red, which reacts primarily with carboxyl groups of acidic sugars. By contrast, in EC, strong staining by ruthenium red of the entire cell wall, of amorphous structures on the surface of EC and of secretory vesicles was observed only after treatment with NaOH. Scanning electron microscopy revealed the presence of amorphous structures on the entire surface of EC but not of NC. These results suggest the abundance of non-methylesterified pectins and the presence of methylesterified and peripherally located pectins in the cell walls of NC and EC, respectively, as well as the absence, in carrot cultured cells, of any correlation between the calcium bridges of pectins and intercellular attachment. Received: 28 April 1998 / Revision received: 20 September 1998 / Accepted: 27 October 1998     

Comparison of the structural features of apple and citrus pectic substances

Pectic substances were extracted from Alcohol Insoluble Solids from lemon peel (albedo) and fractionated by ion exchange chromatography and gelfiltration. The pectin molecules contained rhamnose, arabinose, galactose, glucose and galacturonic acid residues; xylose residues were almost absent. Degradation with purified pectolytic enzymes and subsequent gelfiltration of the resulting pectin fragments showed that the neutral sugar side chains were present in ‘hairy regions’ (blocks of neutral sugar side chains). The distribution of the methoxyl groups was studied by HPLC analysis of enzyme-degraded pectins. Some influence of native pectinesterase on the distribution of the methoxyl groups was found. The results are compared with those of similarly extracted and purified apple pectic substances.     

Purification and characterization of a soluble β-1,4-glucan from bean (Phaseolus vulgaris L.)-cultured cells dehabituated to dichlobenil

Bean cells habituated to grow in the presence of dichlobenil exhibited reduced cellulose and hemicellulose content and an increase in pectic polysaccharides. Furthermore, following the extraction of pectins and hemicelluloses, a large amount of neutral sugars was released. These sugars were found to be part of a soluble β-1,4-glucan in a preliminary characterization, as reported by Encina et al. (Physiol Plant 114:182–191, 2002). When habituated cells were subcultured in the absence of the herbicide (dehabituated cells), the release of neutral sugars after the extraction of pectins and hemicelluloses was maintained. In this study, we have isolated a soluble β-1,4-glucan from dehabituated cells by sonication of the wall residue (cellulose fraction) remaining after fractionation. Gel filtration chromatography revealed that its average molecular size was 14 kDa. Digestion of the sample with endocellulase revealed the presence of cellobiose, cellotriose, and cellotetraose. Methylation analysis showed that 4-linked glucose was the most abundant sugar residue, but 4,6-linked glucose, terminal arabinose and 4-linked galactose for xyloglucan, and arabinogalactan were also identified. NMR analysis showed that this 1,4-glucan may be composed of various kinds of substitutions along the glucan backbone together with acetyl groups linked to the OH group of sugar residues. Thus, despite its relatively high molecular mass, the β-glucan remains soluble because of its unique configuration. This is the first time that a glucan with such characteristics has been isolated and described. The discovery of new molecules, as this β-glucan with unique features, may help understand the composition and arrangement of the polymers within plant cell walls, contributing to a better understanding of this complex structure.     

Simultaneous in vivo truncation of pectic side chains

Despite the wide occurrence of pectin in nature only a few source materials have been used to produce commercial pectins. One of the reasons for this is that many plant species contain pectins with high levels of neutral sugar side chains or that are highly substituted with acetyl or other groups. These modifications often prevent gelation, which has been a major functional requirement of commercial pectins until recently. We have previously shown that modification of pectin is possible through heterologous expression of pectin degrading enzymes in planta. To test the effect of simultaneous modification of the two main neutral pectic side chains in pectic rhamnogalacturonan I (RGI), we constitutively expressed two different enzymes in Arabidopsis thaliana that would either modify the galactan or the arabinan side chains, or both side chains simultaneously. Our analysis showed that the simultaneous truncation of arabinan and galactan side chains is achievable and does not severely affect the growth of Arabidopsis thaliana.     

超滤分离和鉴定三种香菇多糖

用热水从香菇子实体中浸提出香菇多糖,采用两种超滤陶瓷膜将粗多糖分级成三部分Le1,Le2和Le3。所有的这三种多糖都由两组分所组成,采用凝胶过滤色谱测定了多糖分子量,13CNMR和IR光谱测定显示多糖Le1为含α糖甙键的多糖,多糖Le3为含β糖甙键的多糖。采用气相色谱法测定了三种多糖的单糖组成,结果显示三种多糖都由葡糖糖,阿拉伯糖,木糖,甘露糖和半乳糖组成,Le1,Le2和Le3中阿拉伯糖、木糖、甘露糖、半乳糖、葡萄糖的摩尔比分别为0.15∶0.52∶1.00∶1.20∶7.20、0.21∶0.68∶1.00∶1.02∶11.56、0.29∶0.42∶1.00∶0.85∶16.20。三种多糖Le1,Le2和Le3的平均分子量分别为4.02×104、2.16×105和8.93×105。     

A protein-bound polysaccharide from the stem bark of Eucommia ulmoides and its anti-complementary effect

Bioactivity-guided fractionation of a hot-water extract from the stem bark of Eucommia ulmoides led to the isolation of a homogeneous polysaccharide EWDS-2, which was identified as a highly branched protein-bound polysaccharide with average molecular weight between 1000 and 2000 kDa, composed of Glc, Gal, Ara, and Rha in the ratio of 2.2:1.0:0.4:0.2, along with traces of Man and 6.55% of protein. The main linkages of the residues of EWDS-2 include terminal, 1,3-linked, 1,4-linked, 1,2,6-linked, 1,3,6-linked Glc; 1,6-linked, 1,2,6-linked, 1,3,4-linked, 1,4,6-linked Gal; 1,5-linked, 1,3,5-linked Ara; terminal and 1,2,5-linked Rha. The bioassay revealed that EWDS-2 inhibits complement activation on both the classic and alternative pathways with CH₅₀ and AP₅₀ values of 282 ± 11 μg/mL and 144 ± 17 μg/mL, respectively. Preliminary mechanism studies indicate that EWDS-2 inhibits the activation of the complement system by interacting with C1q, C1r, C1s, C2, C3, C4, C5, and C9. The results suggested that EWDS-2 could be valuable for the treatment of diseases associated with the excessive activation of the complement system.     

Characterisation of extracellular polysaccharides from suspension cultures of apple (Malus domestica)

The polymers secreted by suspension-cultured apple cells were composed of 85% carbohydrate (76% neutral sugar and 9% uronic acid) and 15% w/w protein. The extracellular polysaccharides (ECPs) contain 23% XG and 59% AGPs. The monosaccharide composition of the ECPs consisted of Gal, Ara, Glc and Xyl, with smaller amounts of Rha, Fuc and Man. Fractionation of the ECPs by anion-exchange chromatography yielded an unbound neutral fraction and a bound acidic fraction. Monosaccharide and linkage compositions of each fraction were determined. The neutral fraction (48% recovered carbohydrate) was composed of xyloglucan (XG; >90 mol%) which was purified by selective precipitation with Fehling’s solution to yield pure XG. The purified XG had a Glc:Xyl:Gal:Fuc ratio of 4.0:2.5:0.8:0.5; the XG was not O-acetylated. The structure of the secreted XG was similar to that extracted from apple-pomace. The acidic fraction (52% recovered carbohydrate) was composed primarily of arabinogalactan-proteins (AGPs) as detected by the β-glucosyl Yariv diffusion test. The AGP had a Gal:Ara ratio of 1.3: 1.0. Minor amounts of arabinan, xylan and mannan were also detected in the ECPs. This study is the first examination of the polysaccharides secreted by apple cells grown in suspension culture.