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NMR characterization of a 4-O-methyl-β-D-glucuronic acid-containing rhamnogalacturonan from yellow mustard (Sinapis alba L.) mucilage
... Chemical shifts (ı) are expressed in ppm relative to acetone, at ı 30.2. Two-dimensional spectra (COSY and HMQC) were recorded using standard Bruker procedures (Cui, Eskin, Biliaderis, & Marat, 1996). ...
... respectively. That at ı 1.22 ppm was from H-6 of CH 3 groups of the rhamnopyranosyl units (Cui et al., 1996;Habibi, Mahrouz, & Vignon, 2005). ...
... Compared with these components, ␣-galacturonic acid and ␣rhamnopyranose residues were largely retained, their H-1 signals appearing at ı 5.00 and 5.25, respectively, and signals arising from H-6 of CH 3 of rhamnopyranosyl units at ı 1.18 and 1.24 (Fig. 2B). These generally appeared as two partially superimposed doublets, due to the presence of two different rhamnose structures, which were respectively those linked to O-2 and others linked to O-2 and O-4 (Cui et al., 1996;Habibi et al., 2005). ...
A type I arabinogalactan (CPP) was isolated from the viscous gum exuded by Cereus peruvinus (Cactaceae). It contained arabinose, galactose, galacturonic acid, and rhamnose in a 15:66:6:13 molar ratio and had Mw 9 × 105 g mol−1. Methylation analysis and 13C NMR spectroscopy indicated that CPP is composed of a (1 → 4)-linked β-d-Galp main-chain with substituents of α-l-Araf at O-2, O-3 and O-6, which are in turn substituted at O-2, O-3, and O-2,3. These are probably linked to O-4 of some rhamnosyl units of a type I rhamnogalacturonan (RG1). The main chain is formed by repeating (1 → 4)-α-d-GalpA-(1 → 2)-α-l-Rhap groups. The methylation analysis suggested the presence of (1 → 3)-linked β-Galp structures, perhaps linked to the (1 → 4)-linked β-d-Galp backbone. CPP significantly inhibited ethanol-induced gastric lesions in rats at an ED50 of 49 mg kg−1, indicating that it has a gastroprotective effect. The gastric lesion inhibition by CPP suggests a potential use of this polysaccharide, or the crude plant extract, in phytotherapy.
... A variety of polysaccharides from marine plants, microbes and high plants have been reported to demonstrate antioxidant properties [4][5][6]. Yellow mustard mucilage has been systematically studied in previous years including chemical composition, linkage and structural characteristics, as well as physicochemical properties [7][8][9][10][11][12]. These studies revealed that water-soluble yellow mustard mucilage (WSM) was mainly composed of pectic polysaccharides and a small portion of -1,4-linked glucose with occasional side groups [11] . ...
... These studies revealed that water-soluble yellow mustard mucilage (WSM) was mainly composed of pectic polysaccharides and a small portion of -1,4-linked glucose with occasional side groups [11] . Yellow mustard mucilage also exhibits superior emulsification properties [9,12]. Eskin et al. [13] reported that WSM has a potent anti-cancer effect in animal models for colon cancer. ...
... The 13 C chemical shifts were assigned according to the HSQC spectrum. The 13 C chemical and 1 H shifts of the →2,4) α-L-Rhap (1→ residue corresponding to the literature values [9,27,28]. 3.6.2.2. Assignment of residue E (→2) α-L-Rhap (1→). ...
... The connective between H-1 and H-2 of the residues could obtain from COSY spectrum (Fig. 2) and the anomeric carbon chemical shifts could obtain from HSQC spectrum. Based on the above information, as well as the previous literature values [9,27,31,37383940, the residues A, B, C, H, I and J were assigned to be ...
A new sulfated acetamido-heteropolysaccharide, HPS4-2A, was obtained by aqueous extraction followed by precipitation with ethanol and fractionation with DEAE column chromatography from Radix Hedysari. It was composed of rhamnose, arabinose, glucose, galactose and 2-acetamido-2-deoxy-D-galactose in the molar ratio of 10.09%: 25.90%: 25.90%: 25.0%: 12.30%. Elemental analysis indicated that HPS4-2A was a sulfated polysaccharide containing small amount of sulfate groups (1.87%). Partial acid hydrolysis, GC, GC-MS, 1D and 2D NMR spectroscopy analysis of the HPS4-2A revealed a predominance of glucose, galactose and 2-acetamido-2-deoxy-D-galactose linked in a highly-branched structure. The molecular weight of HPS4-2A was determined by HPSEC and HPSEC-MALLS. AFM study indicated that HPS4-2A took a highly branched conformation, which in consistent with the result studied by SEC-MALLS. Structural features of HPS4-2A were also investigated by SEM and TEM. Antioxidant assays demonstrated that HPS4-2A possessed of strong DPPH and hydroxyl radicals scavenging activities, suggesting that HPS4-2A could potentially be used as natural antioxidant.
... Assignments for MeGlcA and GlcA were made based on previous analysis of similar structures as well as by 2D NMR analysis (HSQC and COSY). 27,28 ...
Matrix polysaccharides are a diverse group of structurally complex carbohydrates and account for a large portion of the biomass consumed as food or used to produce fuels and materials. Glucuronoxylan and arabinogalactan protein are matrix glycans that have sidechains decorated with 4- O-methyl glucuronosyl residues. Methylation is a key determinant of the physical properties of these wall glycopolymers and consequently affects both their biological function and ability to interact with other wall polymers. Indeed, there is increasing interest in determining the distribution and abundance of methyl-etherified polysaccharides in different plant species, tissues, and developmental stages. There is also a need to understand the mechanisms involved in their biosynthesis. Members of the Domain of Unknown Function (DUF) 579 family have been demonstrated to have a role in the biosynthesis of methyl-etherified glycans. Here we describe methods for the analysis of the 4- O-methyl glucuronic acid moieties that are present in sidechains of arabinogalactan proteins. These methods are then applied toward the analysis of loss-of-function mutants of two DUF579 family members that lack this modification in muro. We also present a procedure to assay DUF579 family members for enzymatic activity in vitro using acceptor oligosaccharides prepared from xylan of loss-of-function mutants. Our approach facilitates the characterization of enzymes that modify glycosyl residues during cell wall synthesis and the structures that they generate.
... MHz NMR spectrometer (INOVA 600NB, Brucker, Germany) (Cui, Eskin, Biliaderis, & Marat, 1996). ...
Two polysaccharide fractions, SGP I-b and SGP II-b, were purified from the fruiting bodies of Suillus granulatus. The assayed relative molecular masses of SGP I-b and SGP II-b were approximately 17.31 and 10.16 kDa, respectively. SGP I-b consisted of fucose, mannose, glucose, and galactose, whereas SGP II-b consisted of rhamnose, fucose, mannose, glucose, and galactose. The structures of SGP I-b and SGP II-b were preliminarily analyzed through methylation analysis and nuclear magnetic resonance spectroscopy. The results of in vitro antioxidant assays revealed that SGP II-b demonstrated good scavenging ability for hydroxyl and DPPH radicals but poor reduction capacity and scavenging for superoxide anions. SGP I-b did not exhibit any significant antioxidant activities. SGP II-b could also significantly increase lymphocyte proliferation in vitro, however, SGP I-b had no significant effect. Suillus granulatus is a wild, edible medicinal fungus distributed in Northeastern China. S. granulatus polysaccharides (SGP) already be prove have special bioactivities. In the current study, SGP were purified and characterized, and the antioxidant activities and immunomodulatory effects were also assayed. Our work will contribute to the utilization of SGP as natural antioxidants and/or immunity-enhancing agents in food and pharmaceutical industries.
... The region of 5e6 ppm is associated with a-anomeric protons; nonetheless, the region of 4e5 ppm is assigned to b-anomeric protons (Cui, 2005). As a result, what conclusion could be validly drawn from the 1 H spectrum of BSG-3a was that the resonances at 5.12 and 5.08 ppm were definitely due to a-L-Rhap and a-D-GalpA residues, respectively (Arbatsky et al., 2010;Agrawal, 1992;Cui, Eskin, Biliaderis, & Marat, 1996;Nie et al., 2013;Vinod et al., 2008). In addition, the signal in the anomeric region of 4.90 ppm was related to the protons of b-L Arap residues (Jahanbin et al., 2011;Vinod et al., 2008). ...
... A rhamnogalacturonan from yellow mustard mucilage was found to have rhamnose, galactose and glucuronic acid. However, the galactose and glucuronic acid were present in side chain (Cui, Eskin, Biliaderis, & Marat, 1996). ...
Dragon fruit is a tropical fruit with good taste. It can bring health benefits to human body. As one of the major bioactive components in this fruit, the polysaccharides might contribute to the health benefits. However, the precise structure information remains unknown. A leading polysaccharide of dragon fruit pulp, DFPP, was purified and identified by NMR and GC-MS. →4-β-D-GlcpA-1→, →6-β-D-Galp-1→ and →4-α-L-Rhap-1→ constituted the backbone and α-L-Araf–1 → 5-α-L-Araf-1→ formed the branch chain. The precise structure was putatively identified as below. The molecular weight was 2.2 × 103 kDa. The structure information of polysaccharides will be helpful to understand this fruit.
... 1997). Aqueous solutions of mustard mucilage exhibit rheological properties similar to xanthan gum such as shear thinning flow behavior at concentrations above 0.3%, weak-gel properties and interacting synergistically with galactomannans (Weber et al, 1974;Cui and Eskin, 1996). At a low concentration, K 0S%, the mucilage exhibits interfacial activity as it substantially reduces the surface tension of water and the interfacial tension between vegetable oil and water (Weber et al, 1974;Cui et al., 1993a). ...
... AAP possessed unique structural feature, i.e. the presence of-linked 4-O-methyl glucuronic acid residues at terminal ends. There are some reports concerning the presence of-d-glucuronic acid and/or its 4-O-methyl derivatives in arabinogalactans ( Cipriani et al., 2006;Cui, Eskin, Biliaderis, & Marat, 1996;Martínez et al., 2003;Tryfona et al., 2010). These reports support our findings about the presence of 4-O-methyl glucuronic acid in type II arabinogalactan. ...
Two polysaccharides abbreviated ANP and AAP were isolated from the young buds of Acanthopanax sciadophylloides. ANP consisted of l-arabinose, d-mannose, d-glucose and d-galactose in a ratio of ca 1.0:2.6:2.5:1.4 and its weight average molecular weight (Mw) was 1.07×10(4). AAP consisted of l-arabinose, d-galactose and 4-O-methyl-d-glucuronic acid in a ratio of ca 5:10:1, and its Mw was estimated to be 8.40×10(4). ANP was suggested to be an acetylated heteropolysaccharide, whereas AAP was speculated to be a type II arabinogalactan on the basis of structural analysis data. Both polysaccharides were found to stimulate NO production and induce the expression of cytokine mRNAs including IL-1β, IL-6, IL-10 and TNF-α on RAW264.7 cells. They also induced NF-κB activation in RAW-Blue cells. NO production and NF-κB activation by both polysaccharides were decreased by pretreatment with neutralizing anti-TLR-4 and anti-CD14 antibodies but not with anti-TLR-2, anti-SR-A, anti-CD11c, and anti-Dectin-1 antibodies. Therefore, these immunostimulating effects of ANP and AAP were suggested to be promoted by the interaction through the membrane receptors, TLR-4 and CD14. In addition to immunomodulating effects, ANP showed anti-HSV-2 effects in vitro.
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... The pectin arabinogalactans are based on either a linear b (1-4) galactan (type I) or a branched b (1-3,6) galactan (type II) core structure. Flax seed (Warrand et al., 2005) and yellow mustard seed (Cui et al., 1996) mucilages provide sources of rhamnogalacturonan, a major fraction from the latter containing type I galactan core structures. Gum tragacanth and gum karaya (Wang, 2000), and Okra mucilage (Vayssade et al., 2010) contain pectin-like rhamnogalacturonan structures, and gum ghatti contains a soluble arabinogalactan (Kang et al., 2010). ...
This review discusses the structure of pectin, the effects of modification procedures used to prepare modified pectins, and the evidence for the bioactivity of these modified materials. It will consider the evidence for the selective binding of pectin fragments to the pro-metastatic regulatory protein galectin-3 (Gal3) and the potential effects of such binding on reducing the risks of the onset and prevention of cancer. The possible modes of uptake and transport by the body of orally consumed pectin will also be considered. In this context the uptake mechanisms for other dietary carbohydrates that show immunomodulating and anti-cancer properties, such as the β-glucans, will be discussed in terms of possible analogous behaviour for pectin. Finally the article will also consider other dietary carbohydrates that might provide a source of inhibitors for Gal3 and the possible roles of Gal3 and other members of the galectin family in cellular and tissue homeostasis that suggest potential roles for pectin-derived components in combating a range of chronic diseases.
... Chemical shifts (d) are expressed in ppm relative to acetone, at d 2.44 and 30.2 (H 3 CCOCH 3 ) respectively. Two-dimensional spectra (HMQC and HMBC) were recorded using standard Bruker procedures (Cui, Eskin, Biliaderis, & Marat 1996). ...
Fructooligosaccharides have been isolated from roots and leaves of Stevia rebaudiana, by hot aqueous extraction, followed by precipitation with ethanol. Their structure has been determined using methylation and NMR analysis, MALDI-TOF, and ESI-MS. Fructooligosaccharides contained almost exclusively (2→1)-linked β-fructofuranosyl, with terminal α-glucopyranosyl and β-fructofuranosyl units. MALDI-TOF and ESI-MS analyses showed the wide range of degree of polymerisation (DP) present in various extracts. From roots and leaves, three different fractions gave profiles of homologous series, with DPs ranging up to 17 with MALDI-TOF and 19 using ESI-MS. These inulin-type fructooligosaccharides were the major component of extracts from S. rebaudiana roots and significant components from the leaves.
... The methylation analysis was carried out according to the method of Ciucanu and Kerek (1984) with a slight modification. Uronic acids were reduced to neutral polysaccharide before methylation following the previous procedure (Cui, Eskin, Biliaderis, & Marat, 1996;Taylor & Conrad, 1972;York, Darvill, McNeil, Stevenson, & Albersheim, 1986), with modifications as described in the earlier parts of this series. GCeMS was performed on an SP-2330 (Supelco, Bellefonte, Pa) capillary column (30 m  0.25 mm, 0.2 m film thickness) equipped with an ion trap MS detector, programmed from 210 to 240 C at 5 C per min. ...
The structure of a globular gum ghatti fraction (FS) with low surface tension was investigated using methylation–GC–MS, 1D (1H, 13C) and 2D (COSY, TOCSY, HMQC and HMBC) NMR spectra. FS was obtained from the supernatant after sequential ethanol precipitation of aqueous gum ghatti (Gatifolia SD) solutions after 80% (v/v) ethanol. FS is proposed to be a highly branched polysaccharide with small amount of acetyl substitution:R can be represented by the following groups: →2,3-Manp1→, →3,4-Glcp1→, →4-Galp1→, T-α-l-Araf 1→, T-GlcpA1→,T-Galp1→ and T-l-Arap 1→. Galactose has the -β-d configuration, while the arabinose and rhamnose are in α-l form.
... It is noted that a clear signal at 59.0 ppm in the NMR of RG-I-2 was observed for the methyl group attached to C-4 of the GlcA (Cui, Eskin, Biliaderis, & Marat, 1996;Pinto, Martinez, Ocando, & Rivas, 2001). Correspondingly, the signals at 176.9 ppm and 102.6 ppm were assigned to the carboxyl groups and C-1 of the terminal non-reducing GlcA, respectively. ...
Five rhamnogalacturonan I (RG-I) domains RG-I-1, RG-I-2, RG-I-3A, RG-I-3B and RG-I-4 were isolated from ginseng pectin by endo-polygalacturonase hydrolysis and a combination of ion-exchange and gel-permeation chromatography. The five domains all contain galacturonic acid, rhamnose, galactose and arabinose as main components and their rhamnose/galacturonic acid is from 0.26 to 0.64, among the range of RG-I. The molecular weights of RG-I-1 (5 kDa), RG-I-2 (4 kDa) and RG-I-3B (6 kDa) are smaller than those of RG-I-3A (45 kDa) and RG-I-4 (60 kDa). 13C NMR spectra of all domains showed RG-I features. RG-I-2 and RG-I-3B contained RG-I domains linked with highly methyl-esterified and acetylated homogalacturonan domains and the side chains probably belonging to type I and type II arabinogalactans, while RG-I-3A and RG-I-4 may have the side chains of type I arabinogalactans. 4-O-methyl-β-d-glucuronic acid residues were present at non-reducing terminals of RG-I-2.
... Other signals assignments had been marked inFig. 3, according to the 2D NMR and previously reported assignment (Cui, Eskin, Biliaderis, & Marat, 1996; Wang et al., 2003). Both 1BII and its carboxyl-reduced derivatives (1BIIR) were methylated. ...
Centella asiatica, is mainly found in the south of Asia and the southern hemisphere. The pectin was extracted from C. asiatica and isolated by anion-exchange and gel-filtration chromatography with TLC and GLC analyses. It contained arabinose, rhamnose, galactose, xylose and galacturonic acid. The structural features were elucidated by partial acid hydrolysis, enzymatic degradation, methylation, carboxyl-reduction, NMR spectroscopy and ESI-MS experiments. The backbone was presumed to contain 1, 4-linked α-d-GalA and 1, 2/1, 2, 4-linked α-l-Rha with RG-I and homogalacturonan (HG) types. The side chains were neutral chains including arabinosyl, arabinogalactosyl and xylosyl chains. Most of the side chains were attached to Rha, while partial residues were presumed to link to GalA. 14% GalA residues in backbone contained acetyl groups. With deacetylation and carboxyl-reduction, the pectin and its degraded product showed immuno-stimulating activity to different extent in vitro. These results indicated that the carboxyl and acetyl groups play important roles in the expression of immunological activity.
... The DEPT-135 spectrum (Fig. 12) of gum kondagogu shows a resonance at d 60.7 ppm characteristic of methyl carbons from methoxyl groups, confirming the presence of 4-O-methyl-glucuronic acid in the structure of the polysaccharide (Delgobo, Gorin, Jones, & Lacomini, 1998;Oliveira et al., 2007). The 4-O-methyl-glucuronic acid residue was reported in the structure of Anadenanthera macrocarpa gum, yellow mustard mucilage, Sterculia apetala gum and Acacia glomerosa (Cui, Eskin, Biliaderis, & Marat, 1996;Leo´n de Pinto, Martı´nez, & Sanabria, 2001;Marvelys, Maritza, Lilian, Leon de Pinto, & Julio, 2006;Oliveira et al., 2007). Additionally, signals were also observed for a-D-glucose (d 92.9 ppm (C-1), d 72.5 ppm (C-2), d 73.8 ppm (C-3), d 70.6 ppm (C-4), d 72.3 ppm (C-5), d 61.6 ppm (C-6) and b-D-glucose (d 96.7 ppm (C-1), d 75.1 ppm (C-2), d 76.7 ppm (C-3), d 70.6 ppm (C-4), d 76.8 ppm (C-5) and d 61.7 ppm (C-6)) in gum kondagogu, similar to the one reported earlier for monosaccharides (Bock & Pedersen, 1983). ...
Gum kondagogu (Cochlospermum gossypium), a tree exudate gum is a plant growing naturally in the forests of India. This gum is yet to be commercially exploited, as the physico-chemical properties of this gum are yet to be characterized. Various physico-chemical methods like scanning electron microscopy (SEM), differential scanning calorimetry (DSC), static light scattering (SLS), viscometry, elemental analysis, inductively coupled plasma atomic emission spectrometry (ICP-AES), X-ray diffraction spectrometry (XRD), Fourier transform infrared spectroscopy (FT-IR), GC–MS, 1D(1H and 13C) and 2D nuclear magnetic resonance (NMR) (double-quantum filtered correlated spectroscopy (DQF COSY), heteronuclear single-quantum coherence (HSQC) and heteronuclear multiple-bond coherence (HMBC)) have been employed to characterize this gum in the present study. SEM analysis suggests that the native gum has irregular particle size, while the deacetylated gum was found to be fibrilar. The weight-average molecular weight (Mw) of native and deacetylated gum was determined to be 8.5×106 and 2.5×107 g/mol, respectively, by SLS, while the intrinsic viscosity [η] of native and deacetylated gum kondagogu was observed to be 32.68±0.23 and 59.34±dl/g, respectively. The glass transition temperature of the gum was observed to be 34.5 °C, by DSC. The XRD pattern of the native gum indicates a completely amorphous structure. Elemental analysis of the gum revealed the contents of carbon, hydrogen, nitrogen and sulfur to be 34.97, 5.58, 0.229 and 0.128 (w/w%), respectively. Gum kondagogu had high content of calcium, potassium and magnesium, and lower concentrations of aluminum, cadmium, cobalt, lead and nickel. The major functional groups identified from FT-IR spectrum include 3431/cm (–OH), 1731/cm (CH3CO–), 1632/cm (–COO–), 1429/cm (–COO–) and 1249/cm (–CH3CO). Analysis of acid-hydrolyzed gum by GC–MS, indicated the presence of rhamnose, galacturonic acid, glucuronic acid, β-d-galactopyranose, α-d-glucose, β-d-glucose, galactose, arabinose, mannose and fructose, while the 1D and 2D NMR, revealed the presence of the following sugar residues and their linkages—(1→2) β-d-Gal p, (1→6)-β-d-Gal p, (1→4) β-d-Glc p A, 4-O-Me-α-d-Glc p A, (1→2) α-l-Rha p and (1→4) α-d-Gal p A.
... Glucuronic acid is also a common component of a number of gums and mucilages, including gums and mucilages structurally related to pectins, where it is also present as terminal nonreducing end residue [12]. It has been detected, in the a [12,13] or b [12,14] anomeric configuration, linked to neutral sugars (Gal or Fuc) and thus forming the extremity of a longer side-chain, itself bound to a RG backbone as in pectins. However, another possibility, hitherto only known in gums, is the presence of GlcA directly linked to the O-3 of GalA residues [12]. ...
Sugar-beet pulp was de-esterified and submitted to 72 h hydrolysis by 0.1 M HCl at 80 degrees C. Oligomers containing a single glucuronic acid (GlcA) moiety in addition to n(>/= 2) repeats of the dimer -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1--> were isolated from the hydrolysate by ion-exchange and gel-permeation. Glycosyl linkage composition analysis and 1H NMR studies indicated that the GlcA was attached to O-3 of a galacturonic acid (GalA) residue, as shown for the two pentamers beta-D-GlcpA-(1-->3)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA-(1-->2)-L-Rhap and alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-[beta-D-GlcpA-(1-->3)]-alpha-D-GalpA-(1-->2)-L-Rhap. Substitution by GlcA was estimated as occurring on one GalA residue out of 72 in the rhamnogalacturonan fraction of the backbone of beet pectins.
... D-Galacturonic acid has also been found in bacterial polysaccharides. 125,126 In several Proteus O-antigens, amino acids are amide-linked to the carboxyl group of -D-galacturonic 127 or glucuronic acid. 128 It has been shown that D-galacturonic acid replaces phosphate residues in the lipid A component of the lipopolysaccharide (LPS) from the bacterium Aquifex pyrophilus. ...
This chapter focuses on the low-molecular weight carbohydrates that can be formally considered as the oxidation products of mono- or oligo-saccharides in which an aldehyde group and/or one or more hydroxyl groups have been oxidized to carbonyl and/or carboxyl groups. Some acids are important natural products, for instance, L-ascorbic acid (vitamin C); others, such as the glycuronic acids, are the constituents of abundant polysaccharides. Examples of commercial importance are ascorbic acid, as one of the major water-soluble antioxidants; salts of gluconic acid such as the magnesium salt used in the pharmaceutical industry; and D-ribono-1,4-lactone, a versatile material used in the synthesis of natural products. The oxidation products are divided into two principal categories,—namely, acids (and lactones) and neutral compounds. Three kinds of sugar acids can be formally obtained from the corresponding aldoses: (1) aldonic acids, produced by the oxidation at C–1 of the aldose; (2) uronic acids, formed by the oxidation of the primary alcohol group of the aldose; and (3) aldaric acids, formed by the oxidation of both the aldehyde and the primary alcohol group. Neutral oxidation products such as dialdoses, glycosuloses, and glycodiuloses are also discussed in detail in the chapter.
... The widespread occurrence of GUS in plants also raises the question of its natural substrates within the plant. In the cell walls from diVerent plant species, rhamnogalacturonan fractions of the pectin backbone have been shown to be composed of galactose, arabinose and -D-glucuronic acid (Edashige and Ishii 1997; Aspinall et al. 1967 Aspinall et al. , 1968a An et al. 1994; Cui et al. 1996; Renard et al. 1999). The acidic pH optima of plant GUS and the existence of -D-glucuronic acid in the pectin component of cell wall suggest the possibility that the site of activity of plant GUS may be the cell wall. ...
The enzyme beta-glucuronidase (GUS) is well characterized in animals and microbes. However, this enzyme is not well studied in plants and is widely assumed to be absent in them. In this study we document the ubiquitous presence of GUS in the model plants Arabidopsis thaliana, Oryza sativa, Nicotiana tabacum and Zea mays and record its expression pattern. The pH of the assay buffer was found to be critical with pH 4.0 being optimum for detection in all the species. GUS in plants appears to be associated with growth. In general, younger regions of the organs showed more GUS activity than the older and more mature tissues. In Brassica juncea roots stained for GUS, intense blue color could be seen in the trichoblast cells and the growing root hair cells as compared to the non-root hair forming epidermal cells or the fully elongated root hairs. Cotton fibers showed high GUS activity during the initial phase of elongation while the seed coat, from which the fibers formed, did not stain for GUS activity. The activity in the fibers disappeared after they were fully elongated. The level of GUS activity increased 2.58 folds in leaf tissues of N. tabacum when cultured in MS medium supplemented with 6-benzylaminopurine, while gibberellic acid enhanced GUS activity 2.9 folds in the inter-nodal regions of rice in 12-h treatment. In addition, elongation of stem, root and root hairs in tobacco seedlings was strongly inhibited by the specific inhibitor of GUS, saccharo-1-4-lactone in a reversible manner. Taken together, these evidences suggest a probable association of plant GUS in cell growth.
Tea polysaccharides (TPS) were analyzed and characterized by HPAEC, GPC, FT-IR, AFM, HSQC, and HMBC NMR as well as methylation. The TPS mainly consisted of neutral (F0, F0.2, and F0.3) and acidic polysaccharides (F0.1) rich in glucuronic acid. Moreover, anti-proliferative activity of the TPS on HeLa cells was investigated. It was found that F0 exhibited the highest inhibition rate (36.7%) with 200 µg/ml treatment for 48 h. Structural characterization revealed that F0 had a backbone chain of (1→4)-β-d-Glcp, (1→6)-α-d-Galp, (1→2)-α-l-Rhap and (1→2,4)-α-l-Rhap residues with (1→6)-α-d-Galp and acetyl group side chains.
Immunostimulatory activity of the flaxseed gum neutral fraction (NFG) was investigated. NFG was characterized as a xylose rich heteroglycan through monosaccharide composition analysis, FT-IR, methylation/GC–MS, and 1D/2D-NMR. NFG stimulated NO production and phagocytic activity of macrophages. Secretion of interleukin-6, interleukin-1β, and tumor necrosis factor-α (254.7 pg/mL, 2.5 ng/mL, and 42.9 pg/mL, respectively) was significantly induced by NFG. Mitogen-activated protein kinases of JNK and P38 were activated by NFG with increased phosphorylation of JNK and P38, while NO production was reduced to 6.05 and 4.42 μM by JNK and P38 inhibitor, respectively. Nuclear factor-κB signaling pathway was also activated by NFG with the suppression of IκBα and up-regulation of phosphorylation of IκBα and nuclear factor-κB P65. Toll like receptor-2 was the molecular target of NFG and responsible for the activation of down-stream signaling pathways. Thus, NFG from flaxseed gum may potentially be used as a natural immunomodulator in functional foods.
Isolation and structural characterization of a low-molecular-weight pectic polysaccharide SHPPB-1 isolated from sunflower heads
All authors
Yin-jun Zhang, Jian-xun Zhang, Feng Cheng, Xue-liang Shen & Zhao Wang
DOI
http://dx.doi.org/10.1080/07328303.2016.1221077
Published online
16 September 2016
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A water-soluble polysaccharide JS-MP-1 was isolated from Korean mulberry fruits Oddi (Morus alba L.). Sugar linkage analysis and NMR data confirmed that it is a rhamnogalacturonan type I (RG I) polymer carrying arabinan and arabinogalactan (AG II) side chains. JS-MP-1 reduced dose-dependently the viability of 3T3-L1 pre-adipocyte cells, significantly stimulated the cleavage of caspases 9 and 3 and poly (ADP-ribose) polymerase (PARP) and decreased the ratio of Bcl-2 to Bax expression level that led to mitochondrial dysfunction and apoptosis in pre-adipocyte cells. The apoptotic death was mediated by stimulation of MAPKs (ERK and p38) signalling pathway. These results suggest that JS-MP-1 is able to reduce the number of fat cells and the mass of adipose tissue via inhibition of pre-adipocyte proliferation and thus JS-MP-1 itself or a crude aqueous Oddi extract containing this polysaccharide can be used as functional ingredient of health-beneficial food supplements for the treatment or prevention of obesity disorders.
To investigate polysaccharide structure from Lonicera japonica, and study its effects on behavior of pancreatic cells, a homogenous polysaccharide, LJ-02-1, was extracted and purified from flowers of Lonicera japonica by DEAE-cellulose and Sephacryl S-200HR column. The molecular weight was estimated to be 54kDa. Monosaccharide composition was determined to be rhamnose, galacturonic acid, galactose and arabinose in the molar ratio of 10.77: 7.88: 15.45: 65.89 by analyzing the PMP derivatives of the monosacchardies from 2M trifluoracetic acid hydrolysis via HPLC. Based on methylation analysis, partial acid hydrolysis, and NMR spectra, the polysaccharide was elucidated to be a rhamnogalacturonan backbone and substituted partly at C-4 of rhamnose. The branches were determined to be T- and 1,4,6-linked β-D-Galp, T- and 1,5-linked α-L-Araf. The polysaccharide might inhibit BxPC-3 and PANC-1 pancreatic cancer cells growth at the concentration of 1mg/mL with inhibitory ratio of 66.7% and 52.1%, respectively.
Objective: To study the chemical structural features and conformation of polysaccharide HPS4-1A from Hedysarum polybotrys. Methods: A fraction of water-soluble neutral polysaccharide was obtained from H. polybotrys by aqueous extraction followed by precipitation with ethanol. The homogeneous polysaccharide named HPS4-1A was obtained after treated with Savage method and H2O2, and purified with Sephadex G-200 gel filtration chromatography. Then GC, HPGC, GPC-MALLS, element alanalyser, phenol sulfuric acid method, and Bradford method were used to study the physicochemical property of HPS4-1A; The conformation was primarily analyzed with GPC-MALLS method. The connection, main chain and branched-chain structures, and the condition of branching point were studied by methylation, partial acid hydrolysis, and NMR method. Results: The absolute and relative molecular weights of HPS4-1A were 7.386 × 104 and above 6.68 × 105; HPS4-1A was a heteropolysaccharide and consisted of L-rhamnose, L-arabinose, D-glucose, and D-galactose (1:2:1:2). HPS4-1A proved to be a neutral sugar, with 1, 6- and 1,2, 6-α-D-galactopyranosyl and 1, 5- and 1, 3, 5-α-L-arabinofuranosyl residues in backbone, and 1, 4- and 1, 4, 6-α-D-glucopyranosyl and 1, 2- and 1,2, 4-α-L-rhamnofuranosyl residues in branches. Arabinose mainly connected the end of backbone, and glucose and a small quantity of arabinose mainly connected the end of branches. Conclusion: HPS4-1A is a new neutral heteropolysaccharide from H. polybotrys. HPS4-1A has a random coil state conformation with monodisperse mass distribution.
In the present study, structural characterization and antioxidant activity of a fraction (AAP-2A) of polysaccharides from angelica and astragalus (AAP) were investigated. Characteriztion assay showed that AAP-2A had molecular weight (Mw), root-mean square (RMS) radius and polydispersity index (Mw/Mn) of 2.252 × 103 kDa, 28.4 nm and 1.038, respectively. There were infrared characteristic absorption peaks of polysaccharides in FT-IR spectroscopy. AAP-2A was composed of rhamnose (Rha), galactose (Gal), arabinose (Ara) and glucose (Glc) with a molar ratio of 1:2.13:3.22:6.18 in GC analysis. Methylation analysis combined with NMR spectroscopic analysis demonstrated that a preliminary structure of AAP-2A was proposed as follows: 1,3-linked Rhap, 1,3-linked Galp, 1,3-linked Araf, 1,5-linked Araf, 1,3,5-linked Araf, 1,4-linked Glcp and 1,4,6-linked Glcp interspersed with terminal Glcp. AAP-2A exhibited a surface with a sheet-like appearance in scanning electron microscope and stronger antioxidant capacity compared with AAP.
A water-soluble pectic polysaccharide (MP-A40) was isolated and purified from Mosla chinensis Maxim. cv. Jiangxiangru for the first time, with a molecular weight of 32,600 Da. MP-A40 was comprised of 68.63% galacturonic acid and 13.05% neutral sugar. In addition, arabinose, galactose, rhamnose, mannose and glucose composed the neutral sugar in a relative ratio of 4.94, 3.07, 2.13, 1.62 and 1.29% of the dry weight of MP-A40, respectively. Structural characterization of MP-A40 was investigated by methylation analysis and 1D/2D NMR spectroscopy. From the results, the structure of MP-A40 was revealed as follows: 1,4-linked α-d-GalpA and 1,4-linked α-d-GalpA6Me interspersed with rare t-Araf (0.60%), t-Rhap (1.67%) and t-GalpA (10.15%). Esterification assay showed that about 32% of the carboxylic groups in GalA residues existed as methyl ester. In addition, MP-A40 could inhibit the growth of human leukemic cell line K562 and stimulate nitric oxide production from RAW 264.7 macrophages both in dose-dependent manners.
A new water-soluble polysaccharide (PSP) was isolated from the waste of a Sisal (Agave sisalana) plant after the plant's fibers were extracted. The monosaccharide composition analysis showed that PSP consists of galactose, glucose, mannose, rhamnose, arabinose, and galacturonic acid in a molar ratio of 3.0:2.4:1.4:1.0:0.2:0.2. The structure analysis indicated that the PSP backbone chain comprises 1,4-linked β-D-Galp; 1,3-linked β-D-Glcp; 1,3-linked α-D-Manp; and 1,2-linked α-L-Rhap in a ratio of 3:1:1:1. Some of the Glcp, Manp, and Rhap residues in the backbone chain were branched. The in vitro antioxidant activity assay showed that PSP moderately scavenges hydroxyl and DPPH radicals in a dose-dependent manner.
A neutral polysaccharide MP-1 was isolated and purified from Mosla chinensis Maxim. cv. Jiangxiangru. The average molecular weight was 587.53 kDa and it was composed of glucose, galactose, arabinose, mannose and rhamnose in a molar ratio of 16.02:10.01:4.92:4.62:2.23, and trace of fucose. Partial characterization of MP-1 was elucidated by methylation analysis. MP-1 was composed of 1,4-Glcp (31.76%), followed by 1,2-Rhap (17.11%), 1,2,4-Rhap (6.47%), 1,3,4-Galp (5.05%), 1,4-Manp (3.61%), 1,2,4-Manp (2.55%), 1,5-Araf (3.10%) and 1,4,6-Glcp (1.53%). The branching points were located at O-2, O-4 or O-6 with α-Araf (4.01%), α-Rhap (4.31%), α-Fucp (2.39%), β-Galp (11.02%) and β-Glcp (4.04%) as terminal residues. MP-1 exhibited a dose-dependent enhancement in antioxidant assays in this study. Immunomodulatory effects of MP-1 were assessed by splenocyte proliferation test and nitric oxide production of mouse peritoneal macrophages.
The structure of a high molecular weight heteropolysaccharide (60P) isolated from Artemisia sphaerocephala Krasch was elucidated using methylation analysis and 2D NMR spectroscopy. 60P (Mw: 551kDa) was isolated from A. sphaerocephala Krasch (ASK) seeds, using hot water extraction and ammonium sulphate salt precipitation. Methylation and GC–MS analysis indicated that 60P was mainly composed of three types of sugar residues: 4-Xylp (27.8%), 2,4-Xylp (26.0%) and T-GlcpA (22.5%); small amount of 4-GalpA, 4-Glcp, T-Araf, T-Arap and 4, 6-GalpA were also detected with comparable molar ratio. Based on the results of monosaccharide composition, methylation analysis and NMR spectroscopy, a proposed structure is shown below:R represents one of the following groups: T-Araf, T-Arap, T-Araf→3-Araf (1→ and T-Arap→3-Araf (1→.
The analysis of the uronic acids within crude pectin extracts from banana peel and pineapple flesh, by a modified meta-hydroxydiphenyl assay in the presence and absence of sodium tetraborate, revealed a non-negligible amount of glucuronic acid (4.1–7.2g/100g) together with a much higher quantity of galacturonic acid (37.5–42.8g/100g). This suggested that fragments of glucuronic acid-containing heteroxylans, notably glucuronoarabinoxylans (GAXs), accompanied pectins extraction. A step-wise purification process was therefore developed to efficaciously remove, from pectin extracts, GAX contaminants including non-pectin-incorporated neutral sugar (NS) components. It primarily consisted in treating dialysed pectin extracts or not with 78–85% acidified methanol, which led to the precipitation of pectin-rich fractions while the bulk of GAX and free NS materials were solubilised in the aqueous acid–methanol mixture. Subsequently, advantage was taken of the multivalent ion-binding selectivity and ‘exclusivity’ of the pectin polymer anionic (carboxyl) groups to produce GAX and NS oligosaccharide contaminants-free final isolates, and hence greatly enriched in pectin components.
A new water-soluble polysaccharide (SP1) with molecular weight of 9192Da was isolated from the residue after extracting fiber from sisal leaf and its structure was investigated. Monosaccharide composition analysis showed that SP1 was composed of Rha, GalA, Gal, Ara and Glc in a molar ratio of 2.06:1.92:1.00:0.23:0.15. The structure analysis indicated that the SP1 was comprised of a backbone of alternating 1,2-linked-α-L-Rhap and 1,4-linked-α-D-GalpA units, and the neutral polysaccharide side chains composed of Galp, Ara and Glcp residues were attached to the O-4 position of 1,2-linked-α-L-Rhap residues. SP1 could stimulate ConA-induced T lymphocyte proliferation and had noticeable DPPH radical-scavenging ability.
The chemical structure of a water-soluble pectic polysaccharide (sterculia APS) isolated from boat-fruited sterculia seeds (Semen Sterculiae Lychnophorae) was elucidated using partial acid hydrolysis, methylation analysis and 1D/2D nuclear magnetic resonance (NMR) spectroscopy. Partial acid hydrolysis results revealed that sterculia APS contained galacturonic acids and rhamnose units in the backbone, and arabinose, galactose and xylose residues in the branched chains. Combined the methylation analysis results with NMR spectroscopy, a possible structure of APS was proposed as follow: the backbone consisted of the homogalacturonan (HG) named "smooth" regions by linear linkage of 1,4-α-D-GalpA, and the rhamnogalacturonan-I (RG-I) called "hairy" ones by the linkage of →4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→ partially substituted at O-4 of rhamnose units. The GalpA residues were partially methyl esterified and O-acetylated on C-2 and/or C-3. The side chains were mainly composed of araban and arabinogalactan by the linkages of T-, 1,3-, 1,5-L-Araf and T-, 1,4-, 1,6-, 1,3,6-, 1,3,4-D-Galp, attached to O-4 of the backbone rhamnose units.
The structure of acidic fraction gum (AFG) from flaxseed hulls was elucidated by methylation analysis and 1D/2D NMR spectroscopy. This acidic fraction was separated from water-soluble flaxseed gum using anion-exchange chromatography. AFG consisted of a rhamnogalacturonan-I (RG-I) backbone that features diglycosyl repeating units, →2)-α-l-Rhap-(1→4)-α-d-GalpA-(1→. Rhamnosyl residues (38.2%) were the most abundant neutral sugar component. It was present mainly as unbranched (16.5%) and branched (19.5%) →2)-α-l-Rhap-(1→ at O-3. Most of its branches were terminated by monosaccharides, α/β-d-Galp-(1→ (19.6%), α-l-Fucp-(1→ (4.5%) or β-d-Xylp-(1→ (3.1%). However, when this branching site was occasionally appended with →4)-α-d-GalpA-(1→ or →2)-α-l-Rhap-(1→, side chains may consist of rhamnogalacturonan-I (RG-I), homorhamnan (HR) or a mixture of both. AFG was highly branched as indicated by its high degree of branching (0.55). A possible structure of AFG was proposed: (HR, RG-I, and HG refer to homorhamnan, rhamnogalacturonan-I, and homogalacturonan, respectively. The locations of HR, RG-I, and HG are interchangeable; (m+n)/(n+i)≈1.5. The substitution rate of R(1) is ∼54%. R(1) is mostly monosaccharide (α/β-d-Galp-(1→, α-l-Fucp-(1→ or β-d-Xylp-(1→). R(1) may also occasionally be a longer side chain with more than two residues beginning with →4)-α-GalpA-(1→ or →2)-α-l-Rhap-(1→, wherein the side-chain structure may be similar to part of the main chain.).
The structure of the pectic polysaccharide (ASP3) isolated from roots of Angelica sinensis (Oliv.) Diels was investigated using partial acid hydrolysis, enzymic digestion combined with methylation analysis, and further supported by 1H and 13C NMR spectroscopy techniques. The results indicated that ASP3 contained a backbone of linear homogalacturonan fragments as “smooth regions” and rhamnogalacturonan fragments as “hairy regions” with repeating unit of [→ 4)-α-d-GalpA-(1 → 2)-α-l-Rhap-(1 →]. A total of 58.8% rhamnopyranose residues in the backbone were substituted at O-4 position by the side chains. The side chains contained mainly β-1,6- and β-1,4-galactopyranan bearing 3,6- and 4,6-substituted β-d-galactopyranose residues as branched points and short α-1,5-arabinofuranan possessing 3,5-substituted α-l-arabinofuranose residues as branching points. In addition, β-1,6-galactopyranan side chains were highly branched with α-1,5-arabinofuranan carrying 3-O-substituents (1,3,6-Gal) and terminated by the α-arabinofuranose residues which form arabinogalactan.
Using enzymic digestion with pectinase, controlled Smith degradation and NMR-spectroscopy, some structural features of the hairy region of pectic polysaccharide termed silenan SV from the aerial part of campion Silene vulgaris (Moench) Garke (Oberna behen (L.) Ikonn) were elucidated.Silenan was subjected to enzymic digestion with pectinase to furnish the polysaccharide fraction (SVP). The contained residues of d-galacturonic acid (43%), arabinose, galactose and rhamnose as main constituents. The backbone of the hairy region of silenan was found to consist of α-1,4-galactopyranosyl uronic acid and 2-O-glycosylated rhamnopyranose residues. The side chains contained linear regions of residues of α-1,5-linked arabinofuranose and β-1,3-, β-1,4-linked galactopyranose. Silenan SV and its fragment SVP were subjected to Smith degradation to give fractions SVS and SVPS. These contain the residues of terminal and 2-substituted α-arabinofuranose as well as residues of terminal, 3-, and 2,3-substituted β-galactopyranose. In addition, NMR-spectral data confirmed that the residues of α-rhamnopyranose 2-O-glycosylated with the residues of α-1,4-galactopyranosyl uronic acid of the backbone occurred in the core of SVPS and, therefore, in the backbone of silenan SV.On the basis of data obtained, the hairy regions of silenan were suggested to contain mainly the linear chains of β-1,3-, β-1,4-galactopyranan and α-1,5-arabinofuranan. The chains of α-1,5-linked arabinofuranose, β-1,3- and β-1,4-linked galactopyranose were shown to be involved in the side chains of the hairy region having branching points at 2,3-substituted β-galactopyranose residues.
A water-soluble acidic arabinogalactan, named HBN with a molecular mass of 5.4×105 Da determined by HPGPC, was obtained from Centella asiatica. HBN contained Ara, Gal, Rha, GalA and Xyl in molar ratios of 1.0:1.9:0.26:0.30:0.15. The acetyl content was estimated to be 2.5%. Using methylation analysis, partial acid hydrolysis, NaIO4 oxidation–Smith degradation, RI, NMR, ESI-MS, HPGPC, pectolyase-treatment methods, the structure of HBN was elucidated. HBN had a core composed of Rha and GalA, with arabinogalactosyl and xylosyl chains were attached to this core. Seventy-six percent Ara residues were located at termini and linked to O-6 of Gal residues. HBN had remarkable immunoenhancing activities on T- and B-lymphocytes in vitro and vivo tests. It increased spleen index and inhibited the level of IgG. With the stimulation of SAC, it enhanced the secretion of IFN-γ, IL-12 and IL-6, and inhibited IL-10. Its derivatives by NaIO4 oxidation–Smith degradation and enzyme-treatment possessed immunological activities in vitro.
The purpose of this study was to isolate and perform chemical analyzes as well as biological testing of pectic material from white cabbage isolated by sequential aqueous ionic solutions (SEQAIS) or a simple pure water extraction (PW). Water extraction was aimed at yielding water-soluble pectins only, while the harsher conditions in SEQAIS aimed at extracting proto pectin as well. The pectic material resulting from the various extraction steps was characterized and tested, in order to determine whether structural and biological activity were influenced through different isolation procedures. The SEQAIS fractions obtained were one water-soluble and six partly water-soluble extracts, whereas PW yielded two water-soluble extracts. Sugar composition analysis, linkage analysis, HPSEC molecular weight distribution, HPAEC and 13C NMR were run to obtain structural characteristics of the extracted material. Both extraction procedures resulted in degradation of pectin. Pectin containing highly methyl esterified GalpA probably underwent β-elimination due to neutral pH during PW, while hydrolysis of Araf occurred in the first step of SEQAIS in 50 mM acetic acid pH 4.5. Water-soluble extracts were tested for complement-fixing activity and acidic extracts with degraded side chains showed reduced activity. Authors suggest that extraction conditions at neutral pH should be used in order to withhold side chain structure and immuno-activity.
NMR fingerprinting of the components of finely divided plant cell walls swelled in DMSO has been recently described. Cell wall gels, produced directly in the NMR tube with perdeutero-dimethylsulfoxide, allowed the acquisition of well resolved/dispersed 2D (13)C-(1)H correlated solution-state NMR spectra of the entire array of wall polymers, without the need for component fractionation. That is, without actual solubilization, and without apparent structural modification beyond that inflicted by the ball milling and ultrasonication steps, satisfactorily interpretable spectra can be acquired that reveal compositional and structural details regarding the polysaccharide and lignin components in the wall. Here, the profiling method has been improved by using a mixture of perdeuterated DMSO and pyridine (4 : 1, v/v). Adding pyridine provided not only easier sample handling because of the better mobility compared to the DMSO-d(6)-only system but also considerably elevated intensities and improved resolution of the NMR spectra due to the enhanced swelling of the cell walls. This modification therefore provides a more rapid method for comparative structural evaluation of plant cell walls than is currently available. We examined loblolly pine (Pinus taeda, a gymnosperm), aspen (Populus tremuloides, an angiosperm), kenaf (Hibiscus cannabinus, an herbaceous plant), and corn (Zea mays L., a grass, i.e., from the Poaceae family). In principle, lignin composition (notably, the syringyl : guaiacyl : p-hydroxyphenyl ratio) can be quantified without the need for lignin isolation. Correlations for p-coumarate units in the corn sample are readily seen, and a variety of the ferulate correlations are also well resolved; ferulates are important components responsible for cell wall cross-linking in grasses. Polysaccharide anomeric correlations were tentatively assigned for each plant sample based on standard samples and various literature data. With the new potential for chemometric analysis using the 2D NMR fingerprint, this gel-state method may provide the basis for an attractive approach to providing a secondary screen for selecting biomass lines and for optimizing biomass processing and conversion efficiencies.
An acidic polysaccharide (ATPS-2) was isolated and purified from coarse green tea. ATPS-2 with peak molecular weight of 4430 Da was composed of rhamnose, arabinose, galactose and galacturonic acid. Structure features of the purified ATPS-2 were investigated by a combination of chemical and instrumental analysis, such as periodate oxidation, Smith degradation, (1)H NMR, (13)C NMR, COSY, HSQC, HMBC and ROESY spectrum. It was found that ATPS-2 was a pectic polysaccharide. The backbone proved to consist of the smooth region of alpha-1,4-D-galactopyranosyluronan blocks interconnected by 1,2-linked rhamnose residues involved in the linear sugar chain. The ramified hairy regions were shown to contain the following of the backbone: -->4)-alpha-D-GalpA-(1 --> 2)-alpha-L-Rhap-(1 --> 4)alpha-D-GalpA-(1--> and the side chains attached to the residues of rhamnopyranose of these fragments. The backbone galacturonic acid residues of ATPS-2 were partially O-methyl esterified and acetylated. The side chains contain linear and branched alpha-L-Araf and beta-D-Galp residues.
A new aromatic triterpene was isolated from the root bark EtOAc extract of Euonymus japonicus Thunb, and its chemical structure was elucidated mainly by analysis of MS and NMR spectra.
The structural characterization of branched rhamnogalacturonans (RGs) requires the availability of methods that selectively cleave the Rhap-(1-->4)-alpha-GalAp linkage and thereby generate oligosaccharide fragments that are suitable for mass spectrometric and NMR spectroscopic analyses. Enzymic cleavage of this linkage is often ineffective, especially in highly branched RGs. Therefore, we have developed an improved chemical fragmentation method based on beta-elimination of esterified 4-linked GalpA residues. At least 85% of the carboxyl groups of the GalA residues in Arabidopsis thaliana seed mucilage RG is esterified using methyl iodide or 3-iodopropanol in Me(2)SO containing 8% water and 1% tetrabutylammonium fluoride. However, beta-elimination fragmentation at pH 7.3 and 120 degrees C is far more extensive with hydroxypropyl-esterified RG than with methyl-esterified RG. The non-reducing 4-deoxy-beta-l-threo-hex-4-enepyranosyluronic acid residue formed by the beta-elimination reaction is completely removed by treatment with aqueous N-bromosuccinimide, thereby simplifying the structural characterization of the chemically generated oligoglycosyl fragments. This newly developed procedure was used to selectively fragment the branched RG from peppergrass seed mucilage. The products were characterized using MALDI-TOF mass spectrometry, glycosyl residue composition analysis, and 1 and 2D NMR spectroscopy. Our data show that the most abundant low-molecular weight fragments contained a backbone rhamnose residue substituted at O-4 with a single sidechain, and suggest that peppergrass seed mucilage RG is composed mainly of the repeating unit 4-O-methyl-alpha-d-GlcpA-(1-->4)-beta-d-Galp-(1-->4)-[-->4)-alpha-d-GalpA-(1-->2)-]-alpha-l-Rhap-(1-->.
S3A was a RG-I pectin isolated from Centella asiatica that contained Rha, Ara, Gal, Glc and GalA in molar ratio of 1.0:0.6:1.5:0.2:1.1 and had been found to have a backbone composed mainly of the disaccharide repeat unit, -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->. Based on methylation analysis, NaIO4 oxidation, partial acid hydrolysis and lithium-treatment, the structural features were elucidated. Side chains of S3A were predominantly linked to O-4 of 1,2,4-linked alpha-L-Rhap. The side chains are comprised of arabinosyl chains, galactosyl chains, arabinogalactosyl chains and short glucosyl chains. A total of 45% Rhap in the backbone was substituted by side chains. The arabinosyl residues were mostly distributed in the arabinosyl side chains. According to the immunological results of S3A and its degraded derivatives, S3A had no immunological activity, but its derivatives had immuno-stimulating activities to some extent.
This study was done to investigate whether white cabbage contained polysaccharides with immunostimulatory activity using the complement-fixing test as an indicator. The main polysaccharide isolated was of pectin nature. Methanolysis and (13)C-NMR showed that the polymers consisted of highly esterified alpha-galactopyranoside (alpha-GalpA), significant amounts of alpha-arabinose furanoside (alpha-Araf), beta-Galp and lesser amounts of rhamnose in the pyranose form (Rhap) and xylose in the pyranose form (Xylp). Linkage analyses showed that the alpha-GalpA residues were mainly 1,4-linked with small amounts of 1,3,4-linkages. The alpha-Araf residues were mainly terminally (t)- and 1,5-linked, whereas beta-Galp was t-, 1,3-, 1,6-, and 1,3,6-linked. Positive Yariv reaction indicated polymers with arabinogalactan type 2 like structures. alpha-Rhap was mainly present as 1,2- and 1,2,4-linked residues and Xylp was t- and 1,4-linked. The molecular weight varied greatly and was from 10 to 150 kDa. Cabbage polymers had biological activity and this complement-fixing activity was greatly affected by hydrolytic removal of Araf from pectic side chains.
Seeds of Sinapis alba Linn. (commonly called yellow or white mustard) and their components have been reported to possess anticancer properties. In this study, we evaluated the efficacy of a novel mucilaginous fraction of mustard seeds in inhibiting colonic preneoplastic changes in animal models of sporadic and obesity-associated colon cancer. In two separate studies, male Sprague-Dawley or female Zucker obese rats, injected with azoxymethane (15 or 10 mg/kg body wt. once a week for 2 weeks, respectively), were fed AIN-93G diets with or without 5% mustard mucilage (MM) (w/w) for 8 weeks. Our aim was to measure the ability to modulate the number of aberrant crypt foci (ACF), putative preneoplastic lesions of the colon. The data were classified into total numbers of ACF and large ACF (crypt multiplicity of 4 or more). We report here that 5% MM significantly (p<0.05) decreased the number of total (approximately 21% inhibition) and large (approximately 50% inhibition) ACF in the colons of Sprague-Dawley rats compared to that in untreated controls. In addition, 5% MM supplemented diet significantly lowered (p<0.05) the number of total (approximately 63% inhibition) and large (approximately 60% inhibition) colonic ACF in Zucker obese rats compared to untreated obese rats, and had no effect on fasting plasma cholesterol or triglyceride levels. These results demonstrate the possible role of MM as a functional food against sporadic and obesity-associated colon cancer, and provide impetus to conduct research to understand the underlying mechanism(s) of action.
Yellow or white mustard (Sinapis alba L.) is unique in the mustard family by containing large amounts of mucilaginous material in the seed coat. This material was shown to exhibit similar rheological properties to xanthan gum such as shear thinning flow behavior and weak gel structure. This review will discuss the synergistic interactions between yellow mustard mucilage (YMM) and galactomannans, particularly locust bean gum (LBG), and its potential food applications. In addition, synergistic interactions between YMM, with or without LBG, on starch paste viscosity and syneresis will also be reviewed. The thickening, texturizing and stabilizing properties of YMM, and its ability to form gels at very low concentration in the presence of LBG, could lead to many food and industrial applications.
The possibilities for the extension of spectroscopy to two dimensions are discussed. Applications to nuclear magnetic resonance are described. The basic theory of two‐dimensional spectroscopy is developed. Numerous possible applications are mentioned and some of them treated in detail, including the elucidation of energy level diagrams, the observation of multiple quantum transitions, and the recording of high‐resolution spectra in inhomogenous magnetic fields. Experimental results are presented for some simple spin systems.
Fractionation of deacetylated Khaya senegalensis gum affords two polysaccharide components, and structural studies on the major component are described. Partial acid-hydrolysis of this polysaccharide gives a mixture of aldobiouronic acids, (2-L-rhamnose D-galactopyranosid)uronic acid, and (4-D-galactose 4-O-methyl-D-glucopyranosid)uronic acid. Hydrolysis of the methylated polysaccharide indicates the presence therein of residues of 2,3,4,6-tetra- and 2,3,6-tri-O-methyl-D-galactose, 3-O-methyl-D-rhamnose, 2,3,4-tri-O-methyl-D-glucuronic acid, and 2,3-di-O-methyl-D-galacturonic acid, and, in smaller amount, residues of 2,3,5-tri-O-methyl-D-arabinose, 2,3,4-tri- and 2,4-di-O-methyl-D-galactose, 2,3,4-tri-O-methyl-D-rhamnose, and 3,4-di-O-methyl-D-glucuronic acid. The structural significance of these results is discussed.
Small strain oscillatory Theological tests were carried out to study the synergistic interactions between yellow mustard mucilage and locust bean gum. Synergistic interactions were observed for blends of locust bean gum and yellow mustard mucilage ranging in ratios from 1:1 to 1:9 at total polymer concentrations of 0·5 and 2·0% (w/w), respectively. The rheological data indicated that the water-soluble fraction of yellow mustard mucilage was responsible for the observed synergistic behavior in the blends. The major component of the water-soluble yellow mustard mucilage consists of a 1,4-linked β-D-glucan backbone chain. This 1,4-linked β-d-glucan backbone chain is believed to adopt a rigid-ordered structure that may be responsible for the synergistic interaction with galacto-mannans.
The water-soluble yellow mustard (Sinapis alba L.) Polysaccharides were separated into CTAB-precipitated and CTAB-soluble polysaccharide fractions by precipitation with 5% CTAB. Each of these materials was subsequently fractionated into five fractions by an ion-exchange (DEAE-cellulose) column eluted with a stepwise gradient of NaCl (0.1-1.0 M) in NaAc buffer (pH 5, 25 mM). Of the 10 subfractions obtained, 2 neutral polysaccharide fractions (WSCP-I, WSCS-I) and an acidic fraction (WSCP-III) were identified as the major components responsible for the pronounced shear thinning behavior of yellow mustard mucilage solutions. The WSCP-I and WSCS-I fractions were mainly composed of a 1,4-linked beta-D-glucan, although WSCP-1 appeared to be more heterogeneous than WSCS-I. The WSCP-III fraction consisted of nonreducing end glucuronic acid (13.3%), 1,4-linked galacturonic acid (13.6%), 1,6-linked galactose (22.9%), and 1,2-linked (11%) and 1,2,4-linked (17%) rhamnose. Gel filtration chromatography revealed differences in the molecular size of the constituent polysaccharides among the fractions.
The water-soluble (WS) fraction of yellow mustard mucilage, exhibiting pronounced shear thinning behaviour in aqueous solutions, was separated into a CTAB-precipitated fraction (WSCP) and a CTAB-soluble fraction (WSCS) by precipitation with 5% CTAB (hexadecyltrimethylammonium bromide) under optimum conditions of complexation of CTAB with acidic polysaccharides. The chemical structure and molecular size distributions of WSCP and WSCS were determined by methylation, 13C NMR spectra and gel filtration chromatography in order to examine their effect on the rheological properties of these materials in solution. The major fraction WSCP (52·0%) was a mixture of a pectic material consisting primarily of galacturonic acid, galactose and rhamnose and a 1,4-linked β-d-glucan. The minor fraction WSCS (34·0%) also was composed of two polysaccharide fractions differeing in their molecular size and consisted mostly of neutral sugars and non-reducing end residues of glucuronic acid. Both WSCP and WSCS contribute to the rheological properties of WS solutions although they have different structures and molecular weight distributions. WSCP exhibited similar rheological behaviour to WS as assessed by both dynamic and steady shear flow measurements. In contrast, the WSCS fraction only showed a steady shear rheological pattern similar to WS.
The detection of NMR spectra of less sensitive nuclei coupled to protons may be significantly unproved by a two-dimensional Fourier transform technique involving a double transfer of polarization. The method is adequate to obtain natural abundance 15N spectra in small sample volumes with a commercial spectrometer
An arabinose-containing heptadecasaccharide, generated by endoglucanase digestion of the xyloglucan secreted by suspension-cultured sycamore cells, was structurally characterized. The structure of the heptadecasaccharide was unambiguously determined through combined data generated by 1H-n.m.r. spectroscopic, fast-atom bombardment mass spectrometric, and glycosyl-composition and glycosyl-linkage analyses of the intact heptadecasaccharide and of oligosaccharide fragments of the heptadecasaccharide. 1D and 2D 1H-n.m.r. analyses provided data for assignment of the anomeric configurations of the glycosidic linkages, as well as information about the glycosylic and linkage compositions of the heptadecasaccharide. F.a.b.-m.s. data provided the molecular weight and supplied critical information about the glycosylic composition and glycosylic sequence of the heptadecasaccharide. The heptadecasaccharide was found to be a combination of previously characterized nona- and hepta-saccharide components of xyloglucan. The nonasaccharide was shown to be glycosidically linked to the heptasaccharide through C-4 and the β-glucosyl residue nearest to the nonreducing end of the heptasaccharide component. An arabinosyl residue was glycosidically linked at C-2 of the same β-glucosyl residue at the non-reducing end of the heptasaccharide component of the heptadecasaccharide. Although the presence of arabinosyl residues in sycamore xyloglucan has been recognized since 1973, the location of the arabinosyl residues had not been ascertained.
As a result of a study of the permethylation of sugars in such dipolar aprotic solvents as methyl sulphoxide, the hitherto accepted role of the CH3SOCH−2 anion is questioned. The HO− and H− ions appear to be the effective basic agents in these methylation reactions. This conclusion suggested a new method for the permethylation of sugars involving methyl iodide, a solid base (NaOH, KOH, or tert-BuOH/NaOH), and methyl sulphoxide. Excellent yields (98 ±2%) of permethylation products were obtained in a remarkably short (6–7 min) reaction time. The non-sugar peaks that appear in gas chromatograms of the products of Hakomori methylation were absent when the new reagent was used.
A water soluble 1,4-linked β-d-glucan isolated from yellow mustard mucilage was characterized by one- and two-dimensional NMR spectroscopy. The complete assignment of the 1H and 13C resonances was obtained with the assistance of a heteronuclear shift correlated experiment. The connectivities within the residue were established through homonuclear correlation (COSY) while NOE correlation confirmed the 1,4-linked backbone chain structure. In addition, O-ethyl and O-propyl groups were found linked at C2, C3 and C6 positions in some of the glucosyl residues along the cellulose-like backbone chain.
An arabinoxylan-rhamnogalacturonan complex, comprised of galacturonic acid, rhamnose, arabinose, xylose, and galactose in the ratios 75.9:4.6:5.2:3.5:5.4 and lesser amounts of other constituents, was dissociated from the water-insoluble matrix of cell walls of Zea mays by xylanase and glucuronoxylanase treatment. The solubilized complex retained its integrity when subjected to a series of separation procedures, and analysis of the sugar components throughout the elution profiles exhibited consistent ratios. The complex was subjected to controlled degradation by pectate lyase and pectin lyase, yielding two components comprised of rhamnose, fucose, arabinose, xylose, galactose, and galacturonic acid in the ratios 10.9:1.5:13.1:16.9:27.7:30.0 and 8.5:1.7:11.8:6.6:17.3:54.0, respectively, in addition to di-, tri-, and tetra-saccharides of galacturonic acid. The non-reducing terminals of the latter were characterized by the presence of 4,5-unsaturated hexuronic acid. The structural features of the two complex fractions were partially characterized.
The structure of an acidic polysaccharide secreted by a Xanthobacter sp. has been investigated by glycosyl-residue and glycosyl-linkage composition analyses, and the characterization of oligoglycosyl fragments of the polysaccharide has been carried out by chemical analyses, 1H-n.m.r. spectroscopy, fast-atom bombardment mass spectrometry, and electron-impact mass spectrometry. The polysaccharide, which contains O-acetyl groups (approximately 5%) that have not been located, has the tetraglycosyl repeating unit 1 and belongs to a group of structurally related polysaccharides synthesized by both Alcaligenes and Pseudomonas species.
2D-N.m.r. methods have been used to determine the composition of a mixture of oligosaccharides obtained by enzymic degradation of the modified hairy (ramified) regions of apple pectin with a new rhamnogalacturonase. The structures of the oligosaccharides were based on the unit alpha-Rhap-(1----4)-alpha-GalA-(1----2)-alpha-Rhap-(1----4)- GalA. A-beta-Galp unit was 4-linked to approximately half of the terminal Rhap residues and to half of the (1----2)-linked Rhap residues. The sample contained a mixture of a tetrasaccharide, two pentasaccharides, and one hexasaccharide.
The three potent anti-complementary polysaccharides, GL-PI, GL-PII, and GL-PIV, isolated from the leaves of Panax ginseng C. A. Meyer, were subjected to base-catalysed beta-elimination in the presence of sodium borodeuteride or enzymic digestion with endo-alpha-D-(1----4)-polygalacturonase. beta-Eliminative degradation of GL-PI and GL-PII each gave neutral (IN and IIN) and acidic (IA and IIA) fractions. Each fraction N consisted of Ara, Rha, Gal, and Glc, whereas each fraction A comprised a large proportion of GalA in addition to Rha, Gal, Glc, and GLcA. Methylation analysis and g.l.c.-m.s. showed that each fraction IN and IIN contained Rha-(1----2)-Rha-ol-1-d, Rha-(1----4)-Rha-ol-1-d, Ara-(1----4)-Rha-ol-1-d, Gal-(1----4)-Rha-ol-1-d, Gal-(1----6)-Gal-ol-1-d, and GlcA-(1----4)-Rha-ol-1-d, and that IA and IIA contained Rha----Rha-ol-1-d, HexA----Rha-ol-1-d, and HexA----Rha----Rha-ol-1-d. Methylation analysis indicated that IN and IIN also contained high-molecular-weight 6-linked galactan and 4-linked glucan, and that IA and IIA consisted mainly of 2-linked Rha, 4-linked GalA, and terminal and 6-linked Gal. IIA contained more 2-linked Rha than IA. Endo-alpha-D-(1----4)-polygalacturonase-mediated digestion of GL-PIV produced a high-molecular-weight fraction (PG-1) which was rich in neutral sugars, fragments of intermediate size (PG-2), and oligosaccharides (PG-3). PG-1 contained a rhamnogalacturonan core, galactan (which mainly comprised terminal, 6-linked, and 4,6-disubstituted Gal), and 4-linked glucans. PG-2 contained (1----4)-linked alpha-galacturonan partially branched at position 2 or 3 and a rhamnogalacturonan core in addition to small proportions of Gal and Glc. PG-3 contained large proportions of oligogalacturonides.
A new method for determination of uronic acids with meta-hydroxydiphenyl is introduced. It is simpler, quicker, more sensitive, and more specific than other methods, and it needs lesser amounts of fluid. It is recommended for determination of acid mucopolysaccharides in biological materials.
The molecular weight of mustard seed araban, determined by sedimentation equilibrium and vapor pressure osmometry, corresponds to a molecule containing about 45 sugar units. Arabans extracted at different pH values, extracted from different batches of resting seeds, and extracted from cotyledons after germination are all homogeneous and similar in the ultracentrifuge and on free solution electrophoresis in borate. These findings confirm that mustard seed araban is not a degradation artifact of a large heteropolysaccharide, and show that the change in araban structure which occurs with germination cannot take place by the simple addition or simple partial removal of arabinose units from a polysaccharide subfraction.
A polysaccharide containing L-rhamnose and L-xylose was isolated from the lipopolysaccharide extracted from the cell walls of the reference strain for Stenotrophomonas (Xanthomonas) maltophilia serogroup 010. By means of NMR studies and methylation analysis, the repeating unit of the polymer was identified as a branched tetrasaccharide of the structure shown. [GRAPHICS] itable for use in HF experiments as described in this article. The reactor has the advantages of multiple independent reaction vessels, means to precisely measure liquid KF, means to add measured volumes of reagents directly to the closed reaction vessels during the course of the solvolysis, and provision for rapid temperature changes and control. A series of experiments exploring the direct transition from HF solvolysis to acetolysis conditions by the addition of acetic acid and acetic anhydride, as an alternative to previously published solvolysis termination schemes, is also described.
The O-specific polysaccharide from Escherichia coli O113 lipopolysaccharide was separated from the core and lipid A by mild acid hydrolysis and purified by GPC. Methylation analysis and 1H and 13C NMR spectroscopic studies of the O-deacetylated polysaccharide allowed the determination of the structure of the pentasaccharide repeating unit of the polysaccharide which can be written as [equation: see text] The position of the O-acetyl groups was not determined.
The polysaccharide moiety of the O83 antigen (lipopolysaccharide, LPS) consists of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and D-glucuronic acid in the molar ratios 1:2:1:1. Methylation analysis of the polysaccharide and derived oligosaccharides as well as one- and two-dimensional 1H and 13C NMR spectroscopy of the polysaccharide at pD 1 and 6 showed that the O83 polysaccharide has the primary structure-->6)-alpha-D-Glc p-(1-->4)-beta-D-Glc pA-(1-->6)-beta-D-Gal p-(1-->4)-beta-D- Gal p-(1-->4)-beta-D-Glc pNAc-(1-->.
A combination of commercially available preparations of Aspergillus niger beta-D-galactosidase, endo-alpha-L-arabinanase, alpha-L-arabinosidase, and endo-beta-D-galactanase has been used to generate oligoglycosyl fragments of the backbone of rhamnogalacturonan I (RG-I) that had been isolated from the walls of suspension-cultured sycamore cells. The backbone-cleaving enzyme, which is present in the beta-D-galactosidase preparation, only fragments the RG-I backbone when many of the neutral oligoglycosyl side chains have been removed by the other exo- and endo- glycanases. The oligosaccharides released from the backbone were separated from the partially fragmented RG-I and then purified, as their oligoglycosyl aldonic acids, by HPAEC-PAD. Those backbone fragments with degrees of polymerization (dp's) between 2 and 11 were characterized using one- and two-dimensional 1H NMR spectroscopy, electrospray mass spectrometry, and glycosyl-residue and glycosyl-linkage composition analyses. Two series of oligoglycosyl fragments were identified. The quantitatively predominant series has the structure alpha-D-GalpA-(1 --> 2)- alpha-L-Rhap-[ --> 4)-alpha-D-GalpA-(1 --> 2)-alpha-L-Rhap-(1 --> ]n-4-D-GalpA, and the quantitatively minor series has the structure alpha-L-Rhap-[ --> 4)-alpha-D-GalpA-(1 --> 2)-alpha-L-Rhap-(1 --> ]n-4-D- GalpA (n = 1-5). Thus, the enzyme preparations contain an alpha-L-rhamnosidase in addition to the endo- rhamnogalacturonase. The products of the endo-rhamnogalacturonase provide additional evidence that the backbone of RG-I is composed of the diglycosyl repeating unit: --> 4)-alpha-D-GalpA-(1 --> 2)-alpha-L-Rhap- (1 -->. The endo-rhamnogalacturonase from the A. niger beta-D-galactosidase preparation and the endo- rhamnogalacturonase secreted by Aspergillus aculeatus [H.A. Schols et al. Carbohydr. Res., 206 (1990) 117-129] have the same substrate specificities and generate similar oligoglycosyl fragments.
The structure of the capsular polysaccharide from Klebsiella type K43 has been investigated using sugar and methylation analysis, uronic acid degradation, and NMR spectroscopy on the native and the O-deacetylated polysaccharide. It is concluded that the polysaccharide is composed of pentasaccharide repeating units with the structure [formula: see text] The polysaccharide contains approximately 0.4 equiv of O-acetyl group per repeating unit, located at a primary position.
NMR studies have been performed on the methyl glycosides of some (1-->6)-linked disaccharides. Observed J5,6pro-R and J5,6pro-S values indicate that, for the 6-substituted D-gluco- and D-galacto-pyranosides, the rotamer distribution around the C-5-C-6 bond deviates somewhat from that observed for the respective unsubstituted monosaccharide glycosides. There is also a difference between 6-O-alpha-D- or 6-O-beta-L- on the one hand and 6-O-beta-D- or 6-O-alpha-L-substituted glycosides on the other, with somewhat larger values for J5,6pro-R for the latter two indicating a higher proportion of the gauche-trans conformer. The glycosylation shifts observed for the signals from the 6-protons in the glycosidic linkage were dependent on the type of anomeric and absolute configuration of the glycosyl group. NOE measurements by irradiation of the anomeric proton indicated that sugars 6-substituted with alpha-D- or beta-L-glycosyl groups have highly populated conformations in which H-1 and H-6pro-S are proximal, and for beta-D- and alpha-L-glycosyl groups conformations in which H-1 and H-6pro-R are proximal.
Rhamnogalacturonan I (RG-I), a pectic polysaccharide isolated from the walls of suspension-cultured sycamore cells, was shown by glycosyl-residue composition analysis to contain D-glucosyluronic acid (GlcpA) residues (1 mol%) and 4-O-methyl-D-glucosyluronic acid (4-O-Me-GlcpA) residues (0.5 mol%). These monosaccharides were shown, by glycosyl-linkage analysis, to be present in RG-I as terminal nonreducing residues. The glycosyl sequences containing GlcpA and 4-O-Me-GlcpA were determined by structurally characterizing the acidic oligosaccharides released by partial acid hydrolysis of RG-I. Six acidic oligosaccharides were purified by semipreparative high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and characterized by glycosyl-residue and glycosyl-linkage composition analyses, GLC-CIMS, GLC-EIMS, electrospray MS (ESMS), and 1H NMR spectroscopy. We propose that three of the acidic oligosaccharides characterized, 4-O-Me-beta-D-GlcpA-(1-->6)-D-Gal, beta-D-GlcpA-(1-->6)-D-Gal, and beta-D-GlcpA-(1-->4)-D-Gal, originate from the galactosyl-containing side chains of RG-I. The three other acidic oligosaccharides characterized, alpha-D-GalpA-(1-->2)-L-Rha, alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA+ ++-(1-->2)-alpha-L-Rha, and alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA+ ++-(1-->2)-alpha-L- Rhap-(1-->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rha, were generated by partial hydrolysis of the RG-I backbone. No evidence was obtained for the presence of galactosyluronic acid in the side chains of RG-I.(ABSTRACT TRUNCATED AT 250 WORDS)
Digests of modified hairy regions of apple pectin (MHR) obtained after degradation by rhamnogalacturonase (RGase) were analyzed for oligomer composition using high-performance anion-exchange chromatography and pulsed amperometric detection. A series of oligomers which appear to be characteristic of RGase degradation could be recognized. These oligomers were isolated on a preparative scale by size-exclusion chromatography and preparative anion-exchange chromatography and analyzed for sugar composition. 1H NMR spectroscopy showed that the oligomers consisted of between 4 and 9 sugar units with a backbone of alternating rhamnose and galacturonic acid residues, partly substituted with galactose residues linked to C-4 of the rhamnose moiety. The HPLC elution pattern showed that higher oligomers were also formed during incubation with RGase. These have the same basic structure but may contain other sugar units in addition to those given above. The oligomer composition of RGase digests of MHR isolated from apple, pear, leek, onion, carrot, and potato was very similar. Using anion-exchange chromatography to monitor the degradation of MHR at increasing incubation times, it was found that all the oligomers were present from the initial stages of the enzyme reaction and that the ratio between the different oligomers remained constant with time. Implications of these results for the structure of MHR and the mechanism of RGase action are discussed.
1. Methods were developed for the extraction, fractionation and purification of the more soluble polysaccharides of mustard-seed embryos. 2. One of these components was a pure homopolysaccharide, an araban, which was characterized by analysis, optical rotation, chromatography on diethylaminoethylcellulose and electrophoresis; the hydrolysis products of the methylated polysaccharide were isolated and characterized by the formation of crystalline derivatives. From these studies it emerges that mustard-seed araban is very similar to the family of pectic arabans, except that it is more highly branched than usual and contains a proportion of 1-->2-linkages. 3. A survey of the other polysaccharides of mustard seed, both in the embryos and in the seed coats, suggests a predominance of pectic-type polysaccharides.
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