Intestinal Peyer’s patch-immunomodulating glucomannans from rhizomes of Anemarrhena asphodeloides Bunge

During screening for intestinal Peyer’s patch-immunomodulating polysaccharides from plant resources including medicinal herbs, a potent modulating activity was observed in a crude polysaccharide fraction (AS-1) from the rhizome of Anemarrhena asphodeloides Bunge. Oral administration of AS-1 (100 mg/kg/day) to aged BALB/c mice enhanced productions of IL-10, IFN-γ and IL-6 from Peyer’s patch immunocompetent cells, and its oral administration to ovalbumin (OVA)-fed B10.A mice led to significant suppression on induction of OVA-specific IgE in systemic immune system. Further fractionation of the polysaccharides in the crude polysaccharide fraction, AS-1, yielded 4 polysaccharide fractions that were potently active, and contained glucomannans. Treatment of these polysaccharide fractions with endo-β-d-(1 → 4)-mannanase significantly decreased their activities. Mannanase digestion of the active glucomannan gave both long and short hexosyl-oligosaccharides, whereas konjac glucomannan, which was inactive, released short oligosaccharides. Structural analysis indicates that the long oligosaccharides from the active glucomannan contain mannanase-resistant complex structure comprising β-d-Man and β-d-Glc.     

The presence of natural human antibodies reactive against pharmacologically active pectic polysaccharides from herbal medicines

Direct ELISA was performed using normal human sera and human colostrum, to analyse the presence of antibodies which react with pharmacologically active pectic polysaccharides isolated from plants used in traditional Japanese herbal (Kampo) medicine. All sera and colostrum were shown to contain IgM, IgG, IgA and secretory IgA class antibodies which react with the active pectic polysaccharides to different degrees. The reacting IgG antibody in normal human serum recognized the ramified regions (rhamnogalacturonan core with carbohydrate side-chains) of the pharmacologically active pectic polysaccharides as the active sites for complement-activating activity. Correlation analysis indicated that a significant and positive correlation was observed between reactivity with the reacting antibody of IgG class and the degree of complement-activating activity of the active polysaccharides.The reacting IgG class antibody, which was purified from normal human serum by affinity chromatography on bupleuran 2IIc (a pharmacologically active pectic polysaccharide from the roots of Bupleurum falcatum)-immobilized Sepharose, showed cross-reactivity not only with some other pharmacologically active pectic polysaccharides from other medicinal herbs but also with autoantigens such as single-strand DNA, myosin and tublin from mammals.     

Pectic polysaccharides from Chinese herbs: structure and biological activity

Bioactive pectic polysaccharides have been isolated from Chinese herbs, and their structure, activity and modes of action have been studied. Complement-activating pectin from Angelica acutiloba contained a variety of neutral galactosyl chains which attached to the rhamnogalacturonan core (ramified region), and these neutral galactosyl chains were essential for the expression of complement activating activity, but the polygalacturonan moiety modulated the mode of activation by the ramified region. Another pectin-like polysaccharide, bupleuran 2IIb from Bupleurum falcatum showed a potent immune complex clearance-enhancing activity. Bupleuran 2IIb may enhance Fc receptor expression on the macrophage surface via the ramified region as an active site. An anti-ulcer pectin, bupleuran 2IIc from B. falcatum consists mainly of partially branched polygalacturonan in addition to the ramified region and the rhamnogalacturonan II-like region, and the polygalacturonan region may contribute somewhat to the expression of activity. Collectively considered results have indicated that the pharmacological activity of each of the pectic polysaccharides may depend on their fine chemical structure.     

Immunopontentiating and antitumor activities of the purified polysaccharides from Phellodendron chinese SCHNEID

The polysaccharide fractions were isolated and purified from Phellodendron chinese SCHNEID, and antitumor activities were examined at dosages of 2, 5 and 10 mg/100 g. F-7 and F-8 showed the highest tumor inhibitory activities (inhibition ratio 96.4 and 98.2% in 2 mg/100 g), and in dose of 5 mg/100 g, the inhibitory ratios were 95.3 and 97.5%, respectively. Furthermore, 10 mg/100 g of intraperitoneal (i.p.) injection gave 97.3 and 98.7% of inhibition. In oral administration, the inhibitory activities were not markedly observed, indicating that the polysaccharides are directly acting to immune system. Also the polysaccharides increased the number of circulating blood leukocytes and total peritoneal exudate cells. Although implantation of tumor cells greatly decreased the productivity of antibody (antibody-mediated) and T lymphocyte reactivity (delayed-type) as 6.3 from 9.3 and 5.9 from 7.7, represented by the increase of footpad thickness, respectively. The polysaccharides elevated the reactivity of T lymphocyte in tumor-bearing mice, which were rapidly recovered by discontinuance of sample treatments. Especially, F-2, F-5, F-7 and F-8 remarkably recovered the decreased sensitivity. When the effects on thymidylate synthase (TS) and thymidine kinase (TK) activities were determined, TS activities in the F-2 and F-7-treated mice were markedly suppressed to 73.7% and 79.5% of that in the control (p < 0.01), while there was little difference in TK activity with a slight decrease in F-2 only. However, in i.p. injection, TS activities in the F-2, F-5, F-7 and F-8-treated mice were markedly suppressed to 83% to 85% of that in the control (p < 0.01). Furthermore, there were also significant differences in TK activities in F-2, F-5, F-7 and F-8-treated mice (p < 0.05). These results clearly indicated that the i.p. injection is much effective to suppress tumor growth than oral administration.     

Polysaccharides isolated from Açaí fruit induce innate immune responses

The Açaí (Acai) fruit is a popular nutritional supplement that purportedly enhances immune system function. These anecdotal claims are supported by limited studies describing immune responses to the Acai polyphenol fraction. Previously, we characterized γδ T cell responses to both polyphenol and polysaccharide fractions from several plant-derived nutritional supplements. Similar polyphenol and polysaccharide fractions are found in Acai fruit. Thus, we hypothesized that one or both of these fractions could activate γδ T cells. Contrary to previous reports, we did not identify agonist activity in the polyphenol fraction; however, the Acai polysaccharide fraction induced robust γδ T cell stimulatory activity in human, mouse, and bovine PBMC cultures. To characterize the immune response to Acai polysaccharides, we fractionated the crude polysaccharide preparation and tested these fractions for activity in human PBMC cultures. The largest Acai polysaccharides were the most active in vitro as indicated by activation of myeloid and γδ T cells. When delivered in vivo, Acai polysaccharide induced myeloid cell recruitment and IL-12 production. These results define innate immune responses induced by the polysaccharide component of Acai and have implications for the treatment of asthma and infectious disease.     

Bioactive polysaccharides from the stems of the Thai medicinal plant Acanthus ebracteatus: their chemical and physical features

Crude water-soluble polysaccharides were isolated from Acanthus ebracteatus by hot water extraction followed by ethanol precipitation after pre-treatment with 80% ethanol. The crude polysaccharides were separated into neutral and acidic polysaccharides by anion-exchange chromatography. The neutral polysaccharide (A1001) was rich in galactose, 3-O-methylgalactose and arabinose, whereas the acidic polysaccharide (A1002) consisted mainly of galacturonic acid along with rhamnose, arabinose and galactose as minor components indicating a pectin-type polysaccharide with rhamnogalacturonan type I (RG-1) backbone. 3-O-Methylgalactose is also present in the acidic fraction. Both neutral and acidic fractions showed potent effects on the complement system using pectic polysaccharide PM II from Plantago major as a positive control. A small amount of 3-O-methylgalactose present in the pectin seemed to be of importance for activity enhancement in addition to the amount of neutral sugar side chains attached to RG-1. The relationship between chemical structure and effect on the complement system of the isolated polysaccharides is considered in the light of these data. The presence of the rare monosaccharide 3-O-methylgalactose may indicate that this can be used as a chemotaxonomic marker. The traditional way of using this plant as a medical remedy appears to have a scientific basis.     

不同品种苹果采后后熟软化过程中细胞壁多糖的降解

以2种苹果为试材,提取了不同贮藏时期果实的细胞壁物质和8种细胞壁多糖组分,并采用气相色谱法分析了细胞壁多糖组分的单糖组成。结果表明,在贮藏过程中,‘金星’苹果果肉的硬度下降明显,在贮藏第10天前后出现明显的乙烯释放量高峰,而耐贮藏性‘富士’苹果在贮藏期间只释放极少量的乙烯。‘金星’苹果的Na2CO3-1溶性果胶多糖组分的减少尤为显著。这些结果表明,苹果果实Na2CO3-1溶性果胶多糖组分侧链成分的酶降解,是引起苹果细胞壁多糖网络结构的变化,进而导致果实软化的重要原因之一。     

Polysaccharide composition of the fruit juice of Morinda citrifolia (Noni)

An ethanol-insoluble, high molecular weight fraction was collected from the juice of Morinda citrifolia fruit grown in Viet Nam. The fraction is composed primarily of carbohydrate (67% (w/w)). The polysaccharide fraction consists predominantly of GalAp (53.6mol%), Araf (13.6mol%), Galp (17.9mol%) and Rhap (9.5mol%). Glycosyl linkage analysis suggests the polysaccharide fraction contains mostly the pectic polysaccharides, homogalacturonan (4-GalAp), rhamnogalacturonan I (4-GalAp, 2-Rhap, 2,4-Rhap), arabinan (5-Araf, 3,5-Araf, t-Araf), type I arabinogalactan (4-Galp, 3,4-Galp, t-Araf) and beta-glucosyl Yariv-binding type II arabinogalactan (3,6-Galp, t-Araf). Low levels of xyloglucan (4-Glcp, 4,6-Glcp, t-Xylp, t-Fucp), heteroxylan (4-Xylp) and heteromannan (4-Manp) are also present.     

Purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds

The structure and bioactivity of a polysaccharide extracted and purified from a 4M KOH + H3BO3 solution from Prunus dulcis seed cell wall material was studied. Anion-exchange chromatography of the crude extract yielded two sugar-rich fractions: one neutral (A), the other acidic (E). These fractions contain a very similar monosaccharide composition: 5:2:1 for arabinose, uronic acids and xylose, respectively, rhamnose and galactose being present in smaller amounts. As estimated by size-exclusion chromatography, the acidic fraction had an apparent molecular mass of 762 kDa. Methylation analysis (from the crude and fractions A and E), suggests that the polysaccharide is an arabinan-rich pectin. In all cases, the polysaccharides bear the same type of structural Ara moieties with highly branched arabinan-rich pectic polysaccharides. The average relative proportions of the arabinosyl linkages is 3:2:1:1 for T-Araf:(1-->5)-Araf:(1-->3,5)-Araf:(1-->2,3,5)-Araf. The crude polysaccharide extract and fractions A and E induced a murine lymphocyte stimulatory effect, as evaluated by the in vitro and in vivo expression of lymphocyte activation markers and spleen mononuclear cells culture proliferation. The lymphocyte stimulatory effect was stronger on B- than on T-cells. No evidence of cytotoxic effects induced by the polysaccharide fractions was found.     

Pectic polysaccharide breakdown of cell walls in cucumber roots grown with calcium starvation

Pectic polysaccharides from the roots of cucumber (Cucumis sativus L.) grown in liquid culture medium with or without calcium (1 mm CaCl₂) were studied after extraction successively by hot water and Na hexametaphosphate solution. The Ca²⁺ starvation-treatment caused a striking reduction in content of extracted pectic polysaccharide; from an equivalent weight of cell walls, only 33.1% of the control level was extracted from root cell walls of plants cultured under Ca²⁺ deficiency. The extracted pectic polysaccharides were fractionated into neutral and acidic polymers by a DEAE-Sephadex column. The acidic polymers, which represented more than 76% of the yield, appeared to be a major fraction of extracted pectic polysaccharides. The changes of molecular size and glycosyl residue composition of this fraction were compared for the control and Ca²⁺-deprived samples. The results indicate that Ca²⁺ deficiency caused structural changes which could involve both branching pattern and extent of contiguous galacturonosyl units in the water-solubilized pectic polysaccharides. Ca²⁺ starvation also led to a notable decrease in molecular size of the hexametaphosphate-solubilized polysaccharides and, to a lesser extent, of the water-solubilized fraction as well. In addition, polygalacturonase activity in tissue homogenates increased remarkably with the Ca²⁺ deficiency, whereas β-galactosidase activity did not undergo a change. Thus, it appears that one major effect of Ca²⁺ deprivation was to stimulate polygalacturonase activity, an effect which could be involved in the control of the breakdown of pectic polysaccharides in the cell walls.     

Inositol Metabolism in Plants. III. Conversion of Myo-inositol-2-H to Cell Wall Polysaccharides in Sycamore (Acer pseudoplatanus L.) Cell Culture

Prolonged growth of cell cultures of sycamore (Acer pseudoplatanus L.) on agar medium containing myo-inositol-2-(3)H resulted in incorporation of label predominately into uronosyl and pentosyl units of cell wall polysaccharides. Procedures normally used to distinguish between pectic substance and hemicellulose yielded carbohydrate-rich fractions with solubility characteristics ranging from pectic substance to hemicellulose yet the uronic acid and pentose composition of these fractions was decidedly pectic. Galacturonic acid was the only uronic acid present in each fraction. Subfractionation of alkali-soluble (hemicellulosic) polysaccharide by neutralization followed by ethanol precipitation gave 3 fractions, a water-insoluble, an ethanol-insoluble, and an ethanol-soluble fraction, each progressively poorer in galacturonic acid units and progressively richer in arabinose units; all relatively poor in xylose units.Apparently, processes involved in biosynthesis of primary cell wall continued to produce pectic substance during cell enlargement while processes leading to biosynthesis of typically secondary cell wall polysaccharide such as 4-0-methyl glucuronoxylan were not activated.     

Dimeric calcium complexes of arabinan-rich pectic polysaccharides from Olea europaea L. cell walls

The study carried out in this work concerns the pectic polysaccharides of olive cell walls as present in olive pulp and that remained entrapped in the cellulosic residue after sequential extraction of the cell wall material (CWM) with imidazole, carbonate and KOH aqueous solutions. These polymers, obtained after neutralisation and dialysis of an aqueous suspension of the residue (sn-CR fraction), extracted with 4 M KOH, were arabinan-rich pectic polysaccharides. They accounted for 11–19% of the total pectic polysaccharides found in the olive pulp cell walls of fruits collected in two years and in three stages of ripening (green, cherry and black). The analysis by powder X-ray diffraction highlighted the existence, in all sn-CR fractions, of crystalline phases related with the presence of calcium-pectic polysaccharide complexes (CPPC) occurring in an amorphous carbohydrate network. The relative crystallinity of the CPPC varied linearly with the Ca²⁺/GalA molar ratio until a maximum of 0.57. Size-exclusion chromatography showed that sn-CR fractions possessed a bimodal molecular weight distribution. The lower molecular weight fraction of sn-CR (M_w = 70–135 kDa) was independent on the ripening stage of olive fruit, whereas the higher molecular weight fraction showed values of 1.1, 0.6–0.9 and 0.5–0.7 MDa, respectively, for green, cherry and black olives. Treatment of the sn-CR pectic polysaccharides with a 2 M imidazole solution disrupted the CPPC crystalline network showing the loss of low molecular weight galacturonan-rich material during dialysis (12–14 kDa cut off) and the decrease of molecular weight of the polymers to roughly half. These results allowed to infer the presence of oligogalacturonides held within cell walls by calcium ions and that the pectic polysaccharides of sn-CR fraction occurred in olive pulp cell walls as calcium bridged macrodimers.     

A simple procedure for the synthesis of alpha-32P-nucleoside triphosphates.

Chemical analysis of grapefruit (Citrus paradisi) pectic polysaccharides demonstrated that galacturonic acid constitutes 78% by weight of the total carbohydrates found. The remaining 22% was accounted for by a number of sugars which include galactose, glucose, arabinose, xylose, and mannose and, by weight, galactose accounted for almost 50% of the total neutral sugar components found in these pectic polysaccharides. Treatment of pectic polysaccharides with galactose oxidase followed by reduction of oxidized galactose residues with tritiated potassium borohydride resulted in the labeling of pectic polysaccharides. Analysis of the labeled polysaccharides demonstrated that of the total radioactivity incorporated more than 90% was recovered in the galactose residues. These results clearly demonstrate the successful utilization of the galactose oxidase/tritiated potassium borohydride method in labeling plant pectic polysaccharide.     

Ginseng root water-extracted pectic polysaccharides originate from secretory cavities

A range of molecular probes for cell wall polysaccharides has been used to explore the structure and location of water-extracted pectic polysaccharides occurring in fractions isolated from ginseng roots. The LM19 homogalacturonan (HG) epitope was abundant in an HG fraction and analysis of LM19 binding to a rhamnogalacturonan-I (RG-I) rich-fraction indicated that the LM19 epitope is sensitive to acetylation. A specific RG-I epitope (LM16), four arabinogalactan-protein (AGP) epitopes (LM2, LM14, JIM16, MAC207) and an extensin epitope (JIM20) were found to be abundant and co-located in several isolated polysaccharide fractions including an arabinogalactan fraction and two RG-I fractions. Detection of the RG-I, AGP and extensin epitopes identified in isolated polysaccharide fractions in sections of ginseng roots indicated that they were most abundant in secretory cavities found in the cortical regions of ginseng roots. In addition, the immunocytochemical study indicated that polysaccharide epitope masking is a widespread phenomenon in the primary cell walls of ginseng roots.     

Structural and rheological properties of polysaccharides from mango (Mangifera indica L.) pulp

The structure and rheological properties of water-soluble polysaccharides from industrialized mango pulp were investigated. Soluble fraction (SF) 2 was heterogeneous on high performance size exclusion chromatography, giving two peaks as determined by multi-angle laser light scattering and refractive index detectors. The presence of starch in SF2 was demonstrated by a positive iodine reaction and by 13C nuclear magnetic resonance (NMR) spectroscopy. The presence of pectic polysaccharides was shown by a calorimetric method, 13C-NMR spectroscopy and carboxyl reduction. The main pectic polysaccharide was polygalacturonic acid; type I rhamnogalacturonan was also detected. Analysis of the rheological properties of SF2 showed a pseudoplastic behavior up to 3 g x l(-1). 'Creep and recovery' tests and analysis performed under a dynamic state revealed a weak gel character for solutions at concentrations of 15, 20 and 30 g x l(-1).     

Analysis of Herba Asari polysaccharides and their immunological activity

A water-soluble polysaccharide (HA) was extracted from the Herba Asari root. HA was separated into a starch-like glucan fraction (HA1) and a pectin fraction (HA2) using DEAE-cellulose. HA2 was further fractionated into three pectic polysaccharides, HA2-a, HA2-b and HA2-c, using ion-exchange chromatography. NMR and sugar composition analyses demonstrated that HA2-a is an arabinogalactan (AG) and HA2-b and HA2-c are xylogalacturonans (XGA) with AG domains. Lymphocyte proliferation assays showed that both the neutral polysaccharide and acidic polysaccharide were potent B and T cell stimulators that may have two different modes of action.     

Fractionation and characterization of polysaccharides from abaca fibre

Abaca fibre polysaccharides were fractionated into water soluble, pectic, 1% NaOH soluble, hemicellulosic and cellulose fractions by extraction with hot water, dilute hydrochloric acid (pH 1.6), aqueous 1% NaOH and 17.5% NaOH, respectively. Cellulose (60.4–63.6%) and hemicelluloses (20.8%) were the major polysaccharides in abaca fibres. The hot water soluble polysaccharides contained noticeable amounts of pectic substances and a large proportion of neutral polysaccharides. The pectic polysaccharide preparation was enriched in both galacturonic acid and neutral sugars, including xylose, glucose, galactose, arabinose, and rhamnose. Extraction of the fibre with aqueous 1% NaOH produced the hemicellulose–lignin complex, which was enriched in xylose and, to a lesser extent, glucose-, arabinose- and galactose-containing polysaccharides, together with 7.6% associated lignin. Further extraction of the delignified fibre residue with aqueous 17.5%. NaOH removed the hemicellulose fractions, which were strongly enriched in xylose-containing polysaccharides. Besides ferulic and p-coumaric acids, six other phenolic monomers were also detected in the mixtures of alkaline nitrobenzene oxidation of associated lignin in all the polysaccharide fractions. The content of bound lignin in water soluble, pectic, and 1% NaOH soluble polysaccharides (Fractions 1, 2, and 3), isolated directly from the lignified fibres, was 12 times that of the hemicellulosic preparations (Fractions 4 and 5) isolated from the delignified fibre residues.     

Tissue-related changes in methyl-esterification of pectic polysaccharides in cauliflower (Brassica oleracea L. var. botrytis) stems

Pectic substances are a major component of cell walls in vegetable plants and have an important influence on plant food texture. Cauliflower (Brassica oleracea L. var. botrytis) stem sections at different regions of the mature plant stem have been monitored for tissue-related changes in the native pectic polysaccharides. Chemical analysis detected appreciable differences in the degree of methyl-esterification (ME) of pectic polysaccharides. About 65% of galacturonic acid (GalpA) residues were methyl-esterified in floret tissues. Relative ME showed a basipetal decrease, from 94% in the upper stem to 51% in the lower-stem vascular tissues. The decrease was not related to a basipetal increase in glucuronic acid (GlcpA) residues. The monoclonal antibodies, JIM 5 and JIM 7, produced distinct labelling patterns for the relatively low-methyl-esterified and high-methyl-esterified pectin epitopes, respectively. Labelling was related to cell type and tissue location in the stem. Floret cell walls contained epitopes for both JIM 5 and JIM 7 throughout the wall. Stem vascular tissues labelled more strongly with JIM 5. Whereas pith parenchyma in the upper stem labelled more strongly with JIM 7, in the lower-stem pith parenchyma, JIM 5 labelling predominated. Localization of pectic polysaccharide epitopes in cell walls provides an insight into how structural modifications might relate to the textural and nutritional properties of cell walls. Received: 16 August 1997 / Accepted: 20 December 1997     

Characterization of cell wall polysaccharides from the medicinal plant Panax notoginseng

Panax notoginseng is a commonly used medicinal plant in south-western China. Recent studies indicate that wall polysaccharides are responsible for some of the immunostimulatory activity. Fractionation of the P. notoginseng root powder alcohol insoluble residue (AIR) and its compositional analysis enabled us to deduce the polysaccharide and protein composition of the root cell walls. P. notoginseng walls are composed primarily of polysaccharide (approximately 97% w/w) and some protein. The polysaccharides include pectic polysaccharides (neutral Type I 4-galactan (21%), arabinan (5%), acidic rhamnogalacturonan I (RG I, 2%) and homogalacturonan (HGA, 24%), non-cellulosic polysaccharides (heteroxylan, 3%), xyloglucan (XG, 3%) and heteromannan (1%)) and cellulose (24%). The root AIR also contains Type II AG/AGPs (5% w/w) typically associated with the plasma membrane and extracellular matrix. Thus, P. notoginseng roots contain polysaccharides typical of Type I primary cell walls but are distinguished by their very high levels of Type I 4-galactans and low levels of XGs. The major amino acids in the AIR were Leu (14 mol%), Asx (16 mol%), Glx (10 mol%), Ala (9 mol%), Thr (9 mol%) and Val (9 mol%).     

Enzymatic fingerprinting of Arabidopsis pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis (PACE)

Plant cell wall polysaccharides vary in quantity and structure between different organs and during development. However, quantitative analysis of individual polysaccharides remains challenging, and relatively little is known about any such variation in polysaccharides in organs of the model plant Arabidopsis thaliana. We have analysed plant cell wall pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis. By highly specific enzymatic digestion of a polysaccharide in a cell wall preparation, a unique fingerprint of short oligosaccharides was produced. These oligosaccharides gave quantitative and structural information on the original polysaccharide chain. We analysed enzyme-accessible polygalacturonan (PGA), linear β(1,4) galactan and linear α(1,5) arabinan in several organs of Arabidopsis: roots, young leaves, old leaves, lower and upper inflorescence stems, seeds and callus. We found that this PGA constitutes a high proportion of cell wall material (CWM), up to 15% depending on the organ. In all organs, between 60 and 80% of the PGA was highly esterified in a blockwise fashion, and surprisingly, dispersely esterified PGA was hardly detected. We found enzyme-accessible linear galactan and arabinan are both present as a minor polysaccharide in all the organs. The amount of galactan ranged from ~0.04 to 0.25% of CWM, and linear arabinan constituted between 0.015 and 0.1%. Higher levels of galactan correlated with expanding tissues, supporting the hypothesis that this polysaccharide is involved in wall extension. We show by analysis of mur4 that the methods and results presented here also provide a basis for studies of pectic polysaccharides in Arabidopsis mutants.