Structural analysis of a pectic polysaccharide from the leaves of Diospyros kaki

A pectic polysaccharide DL-2A with a molar mass of 8.5 x 10(5), was obtained from the boiling water extract of Diospyros kaki leaves. It had [alpha]20D -21.8 degrees (c 0.22, H2O) and consisted of rhamnose, arabinose, galactose, xylose and galacturonic acid units in the molar ratio of 0.4:3.4:2.4:1.0:0.8, along with traces of glucuronic acid. About 16.7% of galacturonic acid existed as the methyl ester. A combination of linkage analyses, periodate oxidation, partial acid hydrolysis, selective lithium-degraded reaction, ESIMS, 1H- and 13C- NMR spectral analyses revealed its structural features. It was found that DL-2A possessed an alpha-(1-->4)-galacturonan backbone with some insertions of alpha-1,2-Rhap residues. The side-chains of arabino-3,6-galactan were attached to the backbone via O-4 of Rhap residues and O-3 of GalAp residues, while 4-linked xylose residues (forming short linear chains) were directly linked to O-4 of rhamnose residues, not as part of the xylogalacturonan. These novel structural features enlarge the knowledge on the fine structure of pectic substances in the plant kingdom.     

Structural and immunological studies of a pectin and a pectic arabinogalactan from Vernonia kotschyana Sch. Bip. ex Walp. (Asteraceae)

Two polysaccharides, a pectin (Vk100A2b) and a pectic arabinogalactan (Vk100A2a) with mean Mw 2 x 10(4) and 1.15 x 10(6)Da, respectively, were isolated from the dried powdered roots of Vernonia kotschyana Sch. Bip. ex Walp. by hot water extraction followed by fractionation on DEAE-Sepharose fast flow and Sephacryl S-400 HR. The pectin showed low-complement fixation activity and no influence on proliferation of B or T cells, while the pectic arabinogalactan showed a potent, dose-dependent complement fixation activity and a T cell independent induction of B-cell proliferation. Both polysaccharides induced chemotaxis of human macrophages, T cells and NK cells. exo-alpha-L-arabinofuranosidase and exo-beta-D-galactosidase digestion followed by component sugar and methylation analysis indicated that Vk100A2a consisted of a highly branched rhamnogalacturonan core with approximately 50% of the rhamnose 1,2,4-substituted, side chains rich in terminal-, 1,5-linked and 1,3,5-branched arabinose and terminal-, 1,4-, 1,6-linked and 1,3,6-branched galactose. The enzyme resistant part of Vk100A2a still showed strong complement fixating activity, suggesting that this activity may at least in part be expressed by carbohydrate structures present in the enzyme resistant, inner portion of the polymer.     

Characterization of a pectic polysaccharide from the leaves of Diospyros kaki and its modulating activity on lymphocyte proliferation

Pectin is a group of carbohydrate polymers constructing the primary cell walls and the middle lamella of terrestrial plants. Herein, we demonstrated the structure and immunomodulatory activity of the major pectic polysaccharide DL‐3B₂ isolated from the leaves of Diospyros kaki. Based on composition analysis, methylation analysis, two‐step acid hydrolysis, lithium‐mediated selective degradation, ¹³C NMR spectroscopy, and electrospray ionization mass spectrometry, DL‐3B₂ was found to contain an α‐1, 4‐linked galacturonic acid (GalA) backbone with some insertions of α‐1, 2‐linked rhamnose residues. The arabinan‐ and arabinogalactan‐side chains were attached to O‐4 of the rhamnose residues, whereas the linear arabinoxylan was probably linked to O‐3 of the GalA residues. Immunological tests in vitro showed that DL‐3B₂ could help stimulate lipopolysaccharide‐induced B lymphocyte proliferation, but not ConA‐induced T lymphocyte proliferation, and that the arabinose residues play a role in maintaining this immunological activity. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 649–656, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural features of pectic polysaccharides from the skin of Opuntia ficus-indica prickly pear fruits

After removal of the mucilage with water at room temperature, pectic polysaccharides were solubilized from Opuntia ficus-indica fruit skin, by sequential extraction with water at 60 degrees C (WSP) and EDTA solution at 60 degrees C (CSP). Polysaccharides with neutral sugar content of 0.48 and 0.36 mol/mol galacturonic acid residue were obtained, respectively, in the WSP and CSP extracts. These pectic polysaccharides were de-esterified and fractionated by anion-exchange chromatography, yielding for each extract five fractions, which were thereafter purified by size-exclusion chromatography. Two of these purified fractions were characterized by sugar analysis combined with methylation and reduction-methylation analysis. The study was then supported by (1)H and (13)C NMR spectroscopy. The results showed that the water-soluble fraction WSP3 and the EDTA soluble fraction CSP3, consisted of a disaccharide repeating unit -->2)-alpha-l-Rhap-(1-->4)-alpha-d-GalpA-(1--> backbone, with side chains attached to O-4 of the rhamnosyl residues. The side chains contained highly branched alpha-(1-->5)-linked arabinan and short linear beta-(1-->4)-linked galactan.     

Structure and potential immunological activity of a pectin from Centella asiatica (L.) Urban

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.     

Structural characterization and immunological activity of two cold-water extractable polysaccharides from Cistanche deserticola Y. C. Ma

Two major polysaccharide fractions, CDA-1A and CDA-3B, were isolated from the cold-water extract of Cistanche deserticola Y. C. Ma, a holoparasitic plant and a valuable traditional Chinese medicine, using anion-exchange chromatography on DEAE-cellulose and gel-permeation chromatography on Sephacryl S-300 and Sephadex G-150. Their major structural features were elucidated using component and linkage analyses, periodate oxidation, Smith degradation, partial acid hydrolysis, and NMR spectroscopy. The results indicated that CDA-1A is an alpha-(1-->4)-D-glucan with alpha-(1-->6)-linked branches attached to the O-6 of branch points and that CDA-3B is an RG-I polysaccharide containing a typical rhamnogalacturonan backbone and arabinogalactan or arabinan branches. Bioactivity tests showed that CDA-1A is inert for T-cell proliferation stimulation but active for B-cell proliferation, while CDA-3B is potent for the stimulation of both T- and B-cell proliferation.     

An arabinogalactan from the skin of Opuntia ficus-indica prickly pear fruits

The cold-water extract from the skin of Opuntia ficus-indica fruits was fractionated by anion-exchange chromatography. The major fraction, which was purified by size exclusion chromatography, consisted of a polysaccharide composed of galactose and arabinose residues in the ratio 6.3:3.3, with traces of rhamnose, xylose and glucose, but no uronic acid. The results of methylation analysis, supported by (13)C NMR spectroscopy, indicated that this polysaccharide corresponded to an arabinogalactan having a backbone of (1-->4)-linked beta-D-galactopyranosyl residues with 39.5% of these units branched at O-3. The side-groups consisted either of single L-arabinofuranosyl units or L-arabinofuranosyl alpha-(1-->5)-linked disaccharides. This polysaccharide is thus an arabinogalactan that can be classified in the type I of the arabinogalactan family.     

Structure-immunomodulating activity relationships of a pectic arabinogalactan from Vernonia kotschyana Sch. Bip. ex Walp

Structure and immunological characteristics of the pectic arabinogalactan Vk2a (previously reported as Vk100A2a) from the roots of Vernonia kotschyana Sch. Bip. ex Walp. were investigated after enzymatic digestion of the galacturonan moiety and the side chains of the rhamnogalacturonan structure of Vk2a. endo-alpha-D-(1-->4)-Polygalacturonase digestion released the high molecular weight 'hairy region' (Vk2a-HR) and oligogalacturonides. Vk2a-HR consisted of GalA (4-linked) and Rha (2- or 2,4-linked) in a 1:1 ratio, with 60% of Rha branched at C-4. The Rha located in the rhamnogalacturonan core was branched randomly by Gal units. Vk2a-HR was rich in neutral sugars such as Araf 5- (12.2%) and 3,5-substituted (12.8%) and terminally- (14.1%) linked and Gal 4- (13.0%), 3- (0.9%), 6- (2.2%) and 3,6- (1.1%) substituted. Arabinans with chain lengths up to 11 units were identified. Araf residues were attached to C-3 of alpha-L-(1-->5)-Araf chains and to C-4 of Gal residues. Single Gal units and chains of beta-D-(1-->6)-linked galacto di- to penta-saccharides were attached to a beta-D-(1-->3)-galactan core. All the enzyme resistant fractions expressed potent complement fixation and induction of B-cell mitogenic activity, and the present study indicates that there may be several and possibly structurally different active sites involved in the bioactivity of Vk2a. The bioactive sites may be located both in the more peripheral parts of the molecule but also in the inner core of the 'hairy region' or in larger enzyme-resistant chains.     

Structural and immunological properties of arabinogalactan polysaccharides from pollen of timothy grass (Phleum pratense L.)

Extracts from pollen of timothy grass (Phleum pratense L.) contain up to 20% arabinogalactan proteins (AGPs). Separation of the AGP polysaccharide moieties by tryptic digestion, size exclusion chromatography (GPC), and reverse phase HPLC yielded arabinogalactan fractions AG-1 and AG-2 with molecular weights of approximately 15,000 and approximately 60,000Da, respectively. The backbones of both polysaccharides are composed of (1-->6)-linked beta-D-galactopyranosides with beta-D-GlcUAp or 4-O-Me-beta-D-GlcUAp at their terminal ends as revealed by chemical analysis, FT-IR, MALDI-MS, and NMR spectroscopy. AG-1 contains a small number of beta-l-Araf side chains while AG-2 possesses a variety of (1-->3)-linked units, which consist of beta-l-Araf-(1-->, alpha-l-Araf-(1-->3)-beta-l-Araf-(1-->, and alpha-l-Araf-(1-->5)-beta-l-Araf-(1--> as well as a small number of longer arabinogalactan side chains. In contrast to crude pollen extracts, the immunological properties of the arabinogalactan mixture reveal an IgG4 reactivity instead of IgE reactivity. Structural properties of timothy pollen arabinogalactan might thus influence the immune response.     

Rhamnogalacturonan I in Solanum tuberosum tubers contains complex arabinogalactan structures

A rhamnogalacturonan I polysaccharide was isolated from potato (Solanum tuberosum cv. Posmo) tuber cell walls and characterised by enzymatic digestion with an endo-beta-1 --> 4-galactanase and an endo-alpha-1 --> 5-arabinanase, individually or in combination. The reaction products were separated using size-exclusion chromatography and further analysed for monosaccharide composition and presence of epitopes using the LM5 anti-beta-1 --> 4-galactan and LM6 anti-alpha-1 --> 5-arabinan monoclonal antibodies. The analyses point to distinct structural features of potato tuber rhamnogalacturonan I, such as the abundance of beta-1 --> 4-galactan side chains that are poorly substituted with short arabinose-containing side chains, the presence of alpha-1 --> 5-arabinan side chains substituted with beta-1 --> 4-galactan oligomers (degree of polymerisation > 4), and the presence of alpha-1 --> 5-arabinans that resist enzymatic degradation. A synergy between the enzymes was observed towards the degradation of arabinans but not towards the degradation of galactans. The effect of the enzymes on isolated RG I is discussed in relation to documented effects of enzymes heterologously expressed in potato tubers. In addition, a novel and rapid method for the determination of the monosaccharide and uronic acid composition of cell wall polysaccharides using high-performance anion exchange chromatography with pulsed amperometric detection is described.     

Structural studies of the pectic polysaccharide from duckweed Lemna minor L

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

Structural features of an arabinogalactan gum exudates from Spondias dulsis (Anacardiaceae)

The tree Spondias dulcis, located in Venezuela, exudes a light-brown gum. The polysaccharide, isolated from the original gum, contains galactose, arabinose, mannose, rhamnose, glucuronic acid, and its 4-O-methyl derivative. Application of chemical methods, in combination with 1D and 2D NMR spectroscopy afforded interesting structural features of the gum polysaccharide. The unequivocal presence of rhamnose in the polymer structure was confirmed by chemical and spectral data [1H (1.03 ppm); 13C (16.92 ppm)]. Also confirmed was the existence of 3-O- and 6-O-substitutes galactose residues by the spectral data correlations observed in Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Multiple Bond Correlation (HMBC). Also observed were unequivocal resonances for beta-D-glucuronic acid and its 4-O-methyl derivative, and the presence of 3-O-alpha-L-arabinofuranose and 3-O-beta-L-arabinopyranose residues.     

Antitermitic quinones from Diospyros sylvatica

Six quinones were isolated from the chloroform extract of the roots of Diospyros sylvatica and identified as 2-methyl-anthraquinone, plumbagin, diosindigo, diospyrin, isodiospyrin and microphyllone. The effect of the root extract on the orientation and survival of the subterranean termite, Odontotermes obesus was tested. In addition, four of these quinones were tested on the survival of the subterranean termite. In a direct-choice experiment, exposure to an extract-treated filter disc had a significantly repellent effect over the solvent-treated filter disc. The no-choice experiment revealed the toxic property of the extract as well as the tested quinones and showed high mortality of the O. obesus workers after 48 h on forced exposure. The major termiticidal components identified were plumbagin, isodiospyrin and microphyllone while diospyrin was not toxic to termites at the concentration tested. All the quinones are reported for the first time from D. sylvatica.     

Coumaroyl triterpene lactone, phenolic and naphthalene glycoside from stem bark of Diospyros angustifolia

From the ethanolic extract of stem bark of D. angustifolia three new compounds, a coumaroyl triterpene lactone, diospyrosooleanolide (1), a phenolic glycoside, diospyrososide (2) and a naphthalene glycoside, diospyrosonaphthoside (3) were isolated along with five known compounds (4-8). The structures of these compounds were established on the basis of spectroscopic and chemical evidences.     

Antiprotozoal and cytotoxic naphthalene derivatives from Diospyros assimilis

Chemical investigation of the roots of Diospyros assimilis had led to the isolation and characterization of six naphthalene derivatives, two 2-naphthaldehyes, namely 4-hydroxy-3,5-dimethoxy-2-naphthaldehyde 1, 4-hydroxy-5-methoxy-2-naphthaldehye 2, its related isomer 5-hydroxy-4-methoxy-2-naphthaldehyde 3 and three commonly occurring naphthoquinones, diospyrin 4, 8'-hydroxyisodiospyrin 5 and the simple monomer, plumbagin 6. Their chemical structures were established by detailed NMR investigations including 1H and 13C NMR, HSQC, HMBC and NOESY experiments. In addition, the naphthalene derivatives 1-5 were evaluated for their in vitro antiprotozoal activity against protozoan parasites belonging to the genera Trypanosoma, Leishmania and Plasmodium. Among the tested compounds, naphthaldehyde 1 showed moderate inhibition of the growth of the parasites, T. brucei, T. cruzi, L. donovani with IC50 values of 19.82, 12.28 and 38.78 microM and displayed cytotoxicity towards rat skeletal myoblasts (L-6 cells) with IC50 of 174.94 microM, while 2 and 3 were found to be comparatively less active to 1. The dimeric quinones 4 and 5 exhibited good activity against T. brucei and L. donovani with IC50 of 1.12 and 8.82 microM and 12.94 and 16.66 microM respectively.     

Structural studies on hairy region of pectic polysaccharide from campion Silene vulgaris (Oberna behen)

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 -galacturonic acid (43%), arabinose, galactose and rhamnose as main constituents. The backbone of the hairy region of silenan was found to consist of -1,4-galactopyranosyl uronic acid and 2-O-glycosylated rhamnopyranose residues. The side chains contained linear regions of residues of -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 -arabinofuranose as well as residues of terminal, 3-, and 2,3-substituted β-galactopyranose. In addition, NMR-spectral data confirmed that the residues of -rhamnopyranose 2-O-glycosylated with the residues of -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 -1,5-arabinofuranan. The chains of -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.     

An antitumor pectic polysaccharide from Feronia limonia

An acidic heteropolysaccharide has been isolated from the tropical angiosperm Feronia limonia syn. F. elephantum (family: Rutaceae). A partially carboxymethylated α-(1–4) polygalacturonan backbone structure with 2- and 2,4-O-α- -rhamnopyranosyl, 2- and 2,3-O-α- -arabinofuranosyl and 3-, 2,4-and terminal α- -galactopyranosyl bearing side chains has been tentatively assigned. The preliminary study in the murine model showed some significant in vivo Ehrlich ascites carcinoma cell growth inhibition.     

Anti-hypoxia activity of a polysaccharide extracted from the Sipunculus nudus L

A water-soluble polysaccharide, named as SNP, was extracted and fractioned from the body wall of Sipunculus nudus L. by DEAE-Sepharose anion exchange and Sepharose CL-6B column chromatography. The evaluation for anti-hypoxia activity demonstrated that SNP had significant anti-hypoxic activity on normobarie hypoxia, chemical intoxicant hypoxia and acute cerebral ischemia hypoxia models in mice. SNP also enhanced the number of red blood cell count (RBC) and the concentration of hemoglobin (HGB). The structural characteristics of SNP investigated by high performance size exclusion chromatography, Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry indicated that SNP was a homogeneous polysaccharide with a molecular mass of 350 kD and was composed of rhamnose (28%), fucose (16%) and galactose (56%). The results suggested that SNP could be explored as a novel potential anti-hypoxia agent.