Immunological and structural properties of a pectic polymer from Glinus oppositifolius

The aim of this paper was to further elucidate the structure and the immunomodulating properties of the pectic polymer GOA2, previously isolated from Glinus oppositifolius. Enzymatic treatment of GOA2 by endo-alpha-d-(1 --> 4)-polygalacturonase led to the isolation of three pectic subunits, GOA2-I, GOA2-II, and GOA2-III, in addition to oligogalacturonides. GOA2-I was shown to consist of 1,2-linked Rhap and 1,4-linked GalpA in an approximately 1:1 ratio, and NMR-analysis showed that the monomers were linked together in a strictly alternating manner. The galactose units in GOA2-I were found as terminal-, 1,3-, 1,6-, 1,4-, 1,3,4-, and 1,3,6-linked residues, while the arabinofuranosyl existed mainly as terminal- and 1,5-linked units. A rhamnogalacturonan-I type structure was suggested being the predominant part of GOA2-I. According to linkage analysis GOA2-II and GOA2-III contained glycosidic linkages characteristic for rhamnogalacturonan-II type structures. GOA2 was shown by sedimentation velocity in the analytical ultracentrifuge, to have a broad degree of polydispersity with a mode s(20,w) value of approximately 1.9 S, results reinforced by atomic force microscopy measurements. The polydispersity, as manifested by the proportion of material with s(20,w) > 3 S, decreased significantly with enzyme treatment. The abilities of GOA2, GOA2-I, GOA2-II, and GOA2-III to induce the proliferation of B cells, and to exhibit complement fixing activities were tested. In both test systems, GOA2-I showed significantly greater effects compared to its native pectin GOA2. GOA2-I was in addition shown to exhibit a more potent intestinal immune stimulating activity compared to GOA2. The ability of GOA2 to induce secretion of proinflammatory cytokines was examined. Marked upregulations in mRNA for IL-1beta from rat macrophages and IFN-gamma from NK cells were found.     

Hopane-type saponins from Glinus lotoides Linn

Seven hopane-type saponins were isolated from the methanol extract of Glinus lotoides. Six of them were identified as novel compounds and designated as lotoideside A [3-O-beta-D-xylopyranosyl (1-->2)-alpha-L-rhamnopyranosyl-6 alpha-O-beta-D-xylopyranosyl-22-beta-O-beta-D-glucopyranosyl-16 beta-hydroxy hopane (1)], lotoideside B [3-O-beta-D-xylopyranosyl (1-->2)-alpha-L-rhamnopyranosyl-22-beta-O-beta-D-glucopyranosyl-6 alpha,16 beta-dihydroxyhopane (2)], lotoideside C [3-OD-xylopyranosyl-6 alpha-O-beta-D-xylopyranosyl-16 beta-O-beta-D-xylopyranosyl-22 beta-hydroxyhopane (3)], lotoideside D [3-O-beta-D-xylopyranosyl-16 beta-O-alpha-L-arabinopyranosyl-6 alpha,22-beta-dihydroxyhopane (4)], lotoideside E [3-O-beta-D-xylopyranosyl-6 alpha-O-beta-D-xylopyranosyl-16 beta,22-beta-dihydroxyhopane (5)], and lotoideside F [3-O-beta-D-xylopyranosyl-22-beta-O-beta-D-glucopyranosyl-16 beta-hydroxyhopan-6-one (6)]. The known compound succulentoside B (7) was also encountered. Their structures were elucidated on the basis of one-and two-dimensional NMR spectroscopic techniques, ESIMS and chemical evidences.     

Four new immunomodulating steroidal glycosides from the stems of Stephanotis mucronata

Four new C-21 steroidal glycosides, mucronatosides E (1), F (2), G (3), and H (4), were isolated from the stems of Stephanotis mucronata. Two of them had the rare aglycone with a double bond between C-6 and C-7. Their structures were elucidated on the basis of spectroscopic data and chemical evidence. These isolated compounds were assayed for their immunological activities in vitro against concanavalin A (Con A)- and lipopolysaccharide (LPS)-induced proliferation of mice splenocytes. Compounds 2 and 4 showed immunosuppressive activities in a dose-dependent manner, while compounds 1 and 3 possessed immunoenhancing activities.     

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.     

Antitussive and immunomodulating activities of instant coffee arabinogalactan-protein

A low molecular mass arabinogalactan-protein (AGP) composed of galactose and arabinose with a low protein content, isolated from the instant coffee powder of Coffea arabica beans, has been tested on antitussive (in vivo) and immunomodulating (ex vivo) activities. The results of antitussive tests revealed a significant dose dependant cough-suppressive effect of coffee AGP. It was observed 30 or 60 min after AGP administration and its efficacy lasted during the entire experiment course. Immunological tests showed that AGP affected some mediators of immunocompetent cells of immune system as TNF-α, IFN-γ and IL-2 cytokines. It seems that coffee AGP is a good inductor of both pro-inflammatory cytokines TNF-α and IFN-γ, however, less potent in TNF-α induction in comparison with that of β-d-glucan. Evident induction of TNF-α, IL-2 and IFN-γ cytokines, pro-TH1 polarization supports our conclusion about bio-immunological efficacy of AGP with an emphasis on the cellular immunity.     

An extended set of monoclonal antibodies to pectic homogalacturonan

Three novel rat monoclonal antibodies, designated LM18, LM19 and LM20, were isolated from screens for binding to Arabidopsis thaliana seed coat mucilage. The binding of these antibodies to mucilage subject to enzyme and high pH pre-treatments and to a series of model homogalacturonan-rich pectins with defined levels of methyl-esterification indicated their recognition of pectic homogalacturonan epitopes. The binding capacities of these monoclonal antibodies to cell walls in sections of tobacco stem pith parenchyma were also differentially sensitive to equivalent treatments with high pH buffers and pectate lyase. The epitopes bound by these antibodies display some similarities and some differences to the epitopes recognized by the previously isolated and established pectic homogalacturonan probes JIM5 and JIM7.     

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.     

Cytotoxicity and immunomodulating characteristics of labdane diterpenes from Marrubium cylleneum and Marrubium velutinum

From the aerial parts of Marrubium cylleneum, one labdane nor-diterpene has been isolated together with two labdane diterpenes, hitherto not known as natural products. The structures of the isolated compounds were established by means of NMR [(1)H-(1)H-COSY, (1)H-(13)C-HSQC, HMQC-TOCSY, HMBC, NOESY] and MS spectral analyses. Several diterpenoids from M. cylleneum and M. velutinum were tested for their cytotoxic effect against various cancer cell lines and their immunomodulating potential in human peripheral blood mononuclear cells in standard in vitro assays. Our results show a differential cytotoxicity of some compounds as well as their ability to improve selected lymphocyte functions.     

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

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

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

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

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.     

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.     

Isolation, characterization and structural determination of a unique type of arabinogalactan from an immunostimulatory extract of Chlorella pyrenoidosa

An arabinogalactan was isolated from a hot water extract of freeze-dried cells of the green microalga, Chlorella pyrenoidosa. This hot water extract is a proprietary immunomodulator, with the trademark Respondintrade mark (ONC-107). The arabinogalactan was recovered from the ethanol-soluble fraction of the supernatant resulting from a process that involved controlled ethanol precipitation followed by size exclusion chromatography on Sephadex G-100, then Cetavlon precipitation. Sugar analyses, GC-MS data for (S)-2-octyl glycosides, and 1D and 2D NMR experiments established unambiguously that the repeating unit was -->2)-alpha-L-Araf-(1-->3)-[alpha-L-Araf-(1-->4)]-beta-D-Galp-(1-->. This structure does not fit into any of the known classes of arabinogalactans. SEC/MALS experiments gave a molecular mass for the arabinogalactan isolated as 47 +/- 4 kDa but the original structure was probably larger.     

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.     

Three new species and two new combinations in the Aizoaceae from the Western and Northern Cape of South Africa

Three new species of Aizoaceae from the Western Cape are described. Octopoma tanquanum Klak and Vlokia montana Klak are dwarf shrubby or mat-forming succulents which belong to the Ruschieae in the Ruschioideae. Mesembryanthemum knolfonteinense Klak of the Mesembryanthemoideae is a geophyte. Octopoma tanquanum inhabits relatively low lying arid areas within the Tanqua Karoo and the Little Karoo and is thought to be closely allied to the two species of Octopoma found in the Little Karoo. Both V. montana and M. knolfonteinense grow at relatively high altitudes within the fynbos biome. In addition, Ruschia littlewoodii L.Bolus is transferred to Phiambolia, and two new combinations are made in Antimima for Ruschia hexamera L.Bolus and Ruschia radicans L.Bolus.     

Structural features of a pectic arabinogalactan with immunological activity from the leaves of Diospyros kaki

A water-soluble acidic heteroglycan, DL-3Bb, isolated from the leaves of Diospyros kaki, had [alpha](D)(20) -19.9 degrees (c 0.30, water), and contained rhamnose, arabinose, xylose, galactose and galacturonic acid in the molar ratio of 1.0:4.5:0.7:1.5:1.0. About 44% of the galacturonic acid existed as its methyl ester, and O-acetyl groups (approx 5.7%) were also identified. Its molecular weight was determined to be 9.0x10(5) Da by high-performance gel-permeation chromatography. Its structural features were elucidated by a combination of methylation analysis, periodate oxidation, two steps of partial acid hydrolysis, and 1H and 13C NMR spectroscopy and ESI mass spectrometry. The data obtained indicated that DL-3Bb possessed a backbone of a disaccharide of [-->4)-alpha-GalAp-(1-->2)-alpha-Rhap-(1-->], with approx 58.7% substitution at O-4 of the rhamnopyranosyl residues by beta-(1-->4)-linked xylopyranosyl residues, and by beta-(1-->3) and beta-(1-->6)-linked galactopyranosyl (galactan) residues. The side chains were further substituted by arabinofuranosyl residues at O-2 by beta-(1-->4)-linked xylopyranosyl residues and at O-3 by beta-(1-->6)-linked galactopyranosyl residues. Preliminary tests in vitro revealed that it could stimulate LPS-induced B lymphocyte proliferation, but not for ConA-induced T lymphocyte proliferation. It was proposed that the acid-labile arabinofuranosyl residues in the side chains would not be needed for the expression of the enhancement of the immunological activity, and that the presence of GalAp in the backbone has an important, but not crucial effect on the expression of the activity.     

Water-soluble polysaccharides from Salvia officinalis L. possessing immunomodulatory activity

A water-soluble polysaccharide complex (A) composed of galactose (17.9%), 3-O-methyl-galactose (3.0%), glucose (15.5%), mannose (8.3%), arabinose (30.4%), xylose (7.6%), fucose (2.6%), rhamnose (6.7%), and uronic acids (8.0%) has been isolated from the aerial parts of sage (Salvia officinalis L.) by cold water extraction. It showed a broad molecular-mass distribution pattern (Mw approximately 2000-93,000) with a predominance of polymers with Mw< 10,000. Ion-exchange chromatography of A afforded six polymeric fractions (A1-A6) in which arabinogalactans associated with galacturonan and/or rhamnogalacturonan backbones prevail. Sage polysaccharides were examined for their immunomodulatory activity in the comitogenic thymocyte test which is interpreted as being an in vitro correlate of adjuvant activity. The acidic polysaccharide fractions A2, A3 and A4 exhibited the highest mitogenic and comitogenic activities of all fractions tested, and relatively high SI(comit)/SI(mit) ratios approximately 3 indicate potential adjuvant properties of these polysaccharides.     

An Endoreticulatus species from Ocinara lida (Lepidoptera: Bombycidae) in Taiwan

A microsporidium from the Ficus pest, Ocinara lida, in Taiwan is characterized. The taxonomic position of this species was preliminarily determined by sequencing small subunit rRNA gene (SSUrRNA). Analysis of the SSUrRNA sequence indicated that this isolate from O. lida is a member of the genus Endoreticulatus and belongs to the genetic grouping containing other lepidopteran Endoreticulatus species we have analyzed phylogenetically. The taxonomic position of this isolate was also confirmed by the ultrastructural characteristics of this isolate. The congruence between SSUrRNA sequence analysis and ultrastructural characteristics shows that this isolate is more closely related to Endoreticulatus bombycis than to Endoreticulatus schubergi Zw?lfer.     

Some factors affecting production of pectic enzymes by Aspergillus niger

The effects of varying cultural conditions were assessed for the production of pectic enzymes in a strain of Aspergillus niger, isolated from decaying orange fruit. Polygalacturonase and pectinmethylesterase were found to be inducible by polygalacturonic acid and pectin in the medium, respectively. Ammonium sulphate was the best nitrogen source for the production of both enzymes. There were variations in enzyme levels produced in culture filtrates with age of the culture, the highest levels being in 4-day-old cultures. The temperature and pH also had marked effects on the production of pectic enzymes with the best conditions being 40°C and pH 5, respectively. Surface culture technique gave appreciable enzyme yield, while agitation had an inhibitory effect on enzyme production.     

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.