New polysaccharide from Nerium indicum protects neurons via stress kinase signaling pathway.

Most of the polysaccharides purified from Chinese medicinal herbs showed anti-tumor and immune-stimulating effects. However, little is known about their effects on neuroprotection. Our previous study has demonstrated that polysaccharides (J2, J3 and J4) isolated from the flowers of Nerium indicum (Oleander) exert partial protection in cortical neurons stressed by beta-amyloid (Abeta) peptides or deprivation of nutrition from serum. In this study, we have isolated and characterized a new polysaccharide from the flowers of N. indicum (named as J6) and aimed to investigate its neuroprotective effects against Abeta-induced apoptosis. Pretreatment of the polysaccharide J6 significantly decreased the activity of caspase-3 as well as the cytotoxicity triggered by Abeta peptides in a dose-dependent manner. In contrast to the activation of survival signaling such as Akt found in J2, J3 and J4 fractions, neuroprotective effects of J6 markedly inhibited Abeta peptide-stimulated phosphorylation of c-Jun N-terminal kinase (JNK-1) as determined by Western blot analysis. Taken together, the polysaccharide J6 isolated from the flowers of N. indicum can serve as potential neuroprotective agent against neuronal death in Alzheimer's disease and the neuroprotective mechanism may primarily rely on inactivation of JNK signaling pathway.

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.

A beta-D-glucan isolated from the fruiting bodies of Hericium erinaceus and its aqueous conformation.

HEP3, a beta-D-glucan slightly soluble in water, was isolated from the alkaline extract of the fruiting bodies of Hericium erinaceus. Its chemical structure was investigated by methylation analysis, periodate oxidation, Smith degradation and by IR and NMR spectroscopy. It was shown to have a main chain composed of beta-(1-->3)-linked D-glucopyranosyl residues, with single unit glucosyl branches attached to O-6 of every third backbone residue. Viscometry and Congo red reaction indicated that HEP3 has a highly ordered hydrogen-bond dependent conformation in aqueous solution, which collapses in strong alkaline solution.

[Isolation, purification and structural analysis of a polysaccharide MDG-1 from Ophiopogon japonicus].

AIM: To separate and purify the anti-myocardial ischemic polysaccharide fraction with a homogenous molecular weight from Ophiopogon japonicus, then study the chemical structure of the parts. METHODS: Crude polysaccharides were prepared by extracting the tube root fraction of Ophiopogon japonicus with water, then precipitation with ethanol. From the crude polysaccharides, the polysaccharide of MDG-1 was separated and purified using ultrafiltration, DEAE Sepharose FF and Sephadex G-25 column chromatography. Its structure was studied by complete hydrolysis, periodate oxidation, Smith degradation, methylation analysis, 1H NMR and 13C NMR analysis etc. RESULTS AND CONCLUSION: MDG-1 was a water-soluble beta-D-fructosan, containing a backbone composed of Fruf (2 --> 1), and a branch of Fruf (2 --> 6) Fruf (2 --> per average 2. 8 of main chain residues. Mn, Mw and Mp of MDG-1 were 3 400, 4 800 and 5 000, respectively. MDG-1 contains trace of Glc, which maybe connect to its reducing terminal. Molar ratio of Fru and Glc is approximately 35: 1.

Structural investigation of a novel rhamnoglucogalactan isolated from the fruiting bodies of the fungus Hericium erinaceus.

A new heteropolysaccharide (HEP-1) was isolated from the fruiting bodies of Hericium erinaceus. It was estimated to have a molecular weight of 1.8x10(4) da and showed [alpha](D)(20) +129 (c 0.295, H(2)O). HEP-1 is composed of rhamnose, galactose, and glucose in the ratio of 1.19:3.81:1.00. Its structural features were investigated using composition analysis, methylation analysis, partial hydrolysis, and IR and NMR spectroscopy. The results showed that HEP-1 has a (1-->6)-linked alpha-d-galactopyranosyl backbone with branches that are composed of rhamnose and glucose attached to O-2.

Characterization of polysaccharides from the flowers of Nerium indicum and their neuroprotective effects.

Degeneration of neurons is a key problem in Alzheimer's disease (AD) and neuroprotection is a possible way to safeguard neurons from neurodegeneration. Polysaccharides isolated from Chinese medicinal herbs have been investigated extensively for their anti-tumor and immune stimulating effects. Yet, little is known about the effects of polysaccharides in neurons. Recently, two pure polysaccharides isolated from the flowers of Nerium indicum were shown to stimulate proliferation and differentiation of PC12 pheochromocytoma cells, an effect similar to that observed from nerve growth factor. In this notion, it is hypothesized that polysaccharides isolated from the flowers of N. indicum could exhibit beneficial effects in neurons. In this study, we isolated, characterized and investigated two new polysaccharides from the flowers of N. indicum for their neuroprotective effects on neurons against serum-deprivation and beta-amyloid (Abeta) peptide toxicity in primary rat cortical neuronal cultures. Pretreatment of the polysaccharides significantly reduced the number of apoptotic neurons revealed by DAPI staining when neurons were exposed to serum-free medium. Besides, the polysaccharides could also decrease the activity of caspase-3 triggered by Abeta peptides. Western blot analysis indicated that polysaccharides stimulated the phosphorylation of PDK-1 (Serine 241) and Akt (Threonine 308). In conclusion, the polysaccharides J2, J3 and J4 isolated from N. indicum provide a lead for future development of neuroprotective agent against neuronal death in neurodegenerative diseases and the neuroprotective mechanism may primarily rely on activation of Akt survival signaling pathway.

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 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.

Characterization of a rhamnogalacturonan and a xyloglucan from neriumindicum and their activities on PC12 pheochromocytoma cells.

Two polysaccharides, a rhamnogalacturonan (J1) and a xyloglucan (J2), were isolated and purified from the whole flowers of Nerium indicum and characterized by GC, GC-MS, NMR, and ESMS. The rhamnogalacturonan was found to consist of repeating units of a linear trisaccharide with the structure [-->4)-alpha-GalpA(1-->2)alpha-Rhap(1-->4)-alpha-GalpA(1-->4)-](n)(), while the xyloglucan consisted of repeating units of the trisaccharide, which was (1-->4)-linked beta-glucopyranosyl residues as a backbone and substituted at position 6 linked to terminal alpha-xylopyranose side chains. These two polysaccharides were tested on the proliferation and differentiation on PC12 pheochromocytoma cells and found to have effects similar to nerve growth factor (NGF).

Structural characterization of a water-soluble beta-D-glucan from fruiting bodies of Agaricus blazei Murr.

A beta-D-glucan, Ab2-2N, was isolated from the hot-water extract of fruiting bodies of Agaricus blazei Murr by ethanol precipitation, anion-exchange and gel-permeation chromatography. Its structure was investigated by composition analysis, methylation analysis, Smith degradation, mild hydrolysis, and NMR spectroscopy. It contains a (1-->6)-linked beta-D-glucopyranosyl backbone, with one side chain consisting of terminal and 3-substituted beta-D-glucopyranosyl residues attached at O-3 for every three backbone residues.

Purification, characterization, and modification of T lymphocyte-stimulating polysaccharide from spores of Ganoderma lucidum.

The hot-water extract of the spores of Ganoderma lucidum was shown to have a stimulating effect on concanavalin A-induced mitogenic activity of T lymphocytes. Bioassay-guided separation led to the isolation of a polysaccharide with potent T lymphocyte-stimulating activity by ethanol fractionation, anion-exchange, and size-exclusion chromatography. Based on the composition and methylation analyses, periodate oxidation, Smith degradation, and NMR spectroscopy, the native polysaccharide was shown to be a beta-D-(1-->3)-glucan with branches of terminal glucosyl residues substituted at C-6 of the glucose residues in the main chain. The branching ratio is approximately 20%. A series of sulfated or carboxymethylated derivatives were prepared and their structural features were elucidated by chemical and spectral analyses. The solution conformation and T lymphocyte proliferation effect of the glucans before and after derivatization were compared and discussed. The data obtained indicate that the introduction of ionic groups would significantly affect the original conformation of the native glucan in aqueous solution and further affect T lymphocyte-stimulating activity. The triple-helical structure of the glucans, the nature of the ionic groups, and the density of negative charge were considered to be closely related to this activity.

Structural characterization and biological activities of SC4, an acidic polysaccharide from Salvia chinensis.

AIM: To study the chemical characterization and some biological activities of an acidic polysaccharide, named SC4 from Salvia chinensis. METHODS: Chemical and spectral methods were employed to identify the structural characterization of SC4. Effect of SC4 on the proliferation of T- and B-lymphocytes both in vivo and in vitro was measured at various concentrations, and the protective effects of SC4 on PC12 cells against H2O2-induced injury was observed. RESULTS: SC4 was a highly branched polysaccharide with mean molecular weight of 4.5 10(5), composed of Rha, Xyl, Ser, Gal, and GalA in the molar ratio of 1.0 7.0 5.3 1.2 4.2. Methylation analysis and NMR identified the linkages of the residues of SC4. SC4 increased the B-lymphocyte proliferation and spleen weight remarkably while no obvious changes were examined in T-lymphocyte proliferation, thymus weight, and the level of IgG and C3 in blood of mice. Moreover, SC4 enhanced the PC12 cells viability after H2O2 treatment. CONCLUSION: SC4, the acidic polysaccharide with complicated structure, was a B-lymphocyte stimulator and protected PC12 cells at the concentration of 20 mg/L against H2O2-induced injury.

Structural features of immunologically active polysaccharides from Ganoderma lucidum.

Three polysaccharides, two heteroglycans (PL-1 and PL-4) and one glucan (PL-3), were solubilized from the fruit bodies of Ganoderma lucidum and isolated by anion-exchange and gel-filtration chromatography. Their structural features were elucidated by glycosyl residue and glycosyl linkage composition analyses, partial acid hydrolysis, acetolysis, periodate oxidation, 1D and 2D NMR spectroscopy, and ESI-MS experiments. The data obtained indicated that PL-1 had a backbone consisting of 1,4-linked alpha-D-glucopyranosyl residues and 1,6-linked beta-D-galactopyranosyl residues with branches at O-6 of glucose residues and O-2 of galactose residues, composed of terminal glucose, 1,6-linked glucosyl residues and terminal rhamnose. PL-3 was a highly branched glucan composed of 1,3-linked beta-D-glucopyranosyl residues substituted at O-6 with 1,6-linked glucosyl residues. PL-4 was comprised of 1,3-, 1,4-, 1,6-linked beta-D-glucopyranosyl residues and 1,6-linked beta-D-mannopyranosyl residues. These polysaccharides enhanced the proliferation of T- and B-lymphocytes in vitro to varying contents and PL-1 exhibited an immune-stimulating activity in mice.

[Chemical studies on SC3, a polysaccharide from Salvia chinensis].

AIM: To study the chemical structure of SC3, an acidic polysaccharide from Salvia chinesis. METHODS: Based on chemical (including sugar composition analysis, methylation analysis, uronic acid reduction and partial acid hydrolysis) and spectral analysis (IR, NMR, ESI-MS), the structural characterization of SC3 was investigated. RESULTS: SC3 was composed Rha, Ara, Gal and GalA, with its mean molecular weight of 7.7 x 10(4). By means of methylation analysis, partial acid hydrolysis, NMR and ESI-MS spectrum, the linkages and sequence information of SC3 were obtained. CONCLUSION: SC3 is an complicated acidic polysaccharide, obtained for the first time from the plant.

[Structural features of a neutral heteropolysaccharide CPB-4 from Cynanchum paniculatum].

OBJECTIVE: To study the chemical features of CPB-4, a heteropolysaccharide obtained from Cynanchum paniculatum. METHOD: Sugar composition analysis, methylation analysis, partial hydrolysis and carbon-13 nuclear magnetic resonance were used to determine the sugar composition, linkages, main chain, branch chains and branching points. RESULT: CPB-4 is composed of L-arabinose, L-xylose, L-rhamnose and D-galactose in closely molar ratios of 0.8:0.2:0.2:1.0. Its main chain is comprised of 1, 5 linked galactose and side chains are comprised of terminal xylose, terminal arabinose, oligosaccharide of arabinose and oligosaccharide of arabinose, rhamnose and galactose. The branching points are located at C-6 and C-2 of galactose. CONCLUSION: CPB-4 is a new heteropolysaccharide from C. paniculatum.

Structure and Conformation Behavior of a Glucan from Spores of Ganoderma lucidum (Fr.) Karst.

A novel polysaccharide designated as beta-D-glucan LB-NB, [alpha](D)(21) -24.52(0)(c =0.81, H(2)O)and Mr 4.7x10(4), was isolated and purified from a 0.5 mol/L sodium hydroxide extract of the sporoderm-broken spores of Ganoderma lucidum. The results of NMR experiments, total hydrolysis, methylation analysis and Smith degradation of LB-NB showed that it possesses a backbone consisting of(1right curved arrow 3)- beta-D-glucosyl residues, about two out of nine glucosyl residues being substituted at O-6 by single glucosyl groups. The conformational behavior of LB-NB was studied. Measurements by specific rotations and viscometry showed that the glucan LB-NB takes an ordered structure in water or lower concentration(<0.05 mol/L)alkaline solution and dissolves as random coil in higher concentration(>0.1 mol/L)alkaline solution or in dimethyl sulfoxide(Me(2)SO). The ordered conformation melts into flexible chains with the increase of the concentration of alkali or the V(DMSO)(v/v)of H(2)O-Me(2)SO mixture. The former dissociation is reversible and the later is irreversible. The complex-formation with Congo red indicated that the LB-NB takes neither triple-stranded helical nor randomly coiled conformation in neutral or slightly alkaline solution, and most probably, it should contain single helical structure in aqueous solution. According to immunological test, the LB-NB showed remarkable activity of stimulating the proliferation of T-Cells in vitro, giving a good example for Kulicke's statement.

Structure of a New Glucan from Cynanchum panilatum (Bunge) Kitagawa.

A polysaccharide was purified from a traditional Chinese drug, "Xu-Chang-Qing" (Cynanchum panilatum (Bunge) Kitagawa), and its molecular weight was estimated to be 1.5x10(4) by HPLC method. Its specific rotation [alpha](D) was 151.4 deg; (0.96, H(2)O). Sugar composition analysis indicated this polysaccharide was composed only of glucose. Methylation analysis, partially hydrolysis, acetolysis, IR and NMR data showed that its main chain was composed of alpha-D 1,4 linked glucosyl residues and its side chains were composed of alpha-D 1,4 and alpha-D 1,6 linked glucosyl residues. There was one branch located at O-6 of the glucosyl residue in every repeating unit containing five-glucosyl residues.

Preparation and Structural Analysis of Sulfated Derivatives of Lentinan.

A series of sulfated derivatives of lentinan were prepared by the modified Wolfrom method. Sulfates content analysis indicated that the proportion of sulfate substitution depended on the reaction time and the ratio of chlorsulfonic acid and pyridine in the ester reagent. Methylation analysis by Needs method and Hakomori method was proved not to be suitable for the structural analysis of sulfated lentinan. (13)C-NMR data showed that sulfates were located on the carbon-6 in all sulfated derivatives of lentinan showing the higher reactivity of the hydroxyls on C-6 position than those at other position.