•A xylan CTPB1 was discovered from the roots of Cudrania tricuspidata.•Its repeating unit was a branched (1 → 4)-β-d-xylan.•CTPB1 could induce the proliferation of mouse splenocytes in vitro.To discover new immunomodulator in the food industry, a water-soluble polysaccharide, CTPB1 was obtained from roots of Cudrania tricuspidata (Carr.) Bur. The average molecular weight of this polysaccharide was estimated to be 4.0 × 104 Da. Monosaccharide composition analysis indicated that the only neutral sugar present in this polysaccharide was xylose. From methylation analysis and 1H and 13C NMR spectroscopy, the structure of CTPB1 was deduced to be (4-O-methyl-d-glucurono)-d-xylan, with the 4-O-methyl-d-glucopyranosyluronic acid group linked to O-2 of a (1 → 4)-β-d-xylan. The repeating unit of CTPB1 is composed of one non-reducing terminal residue of 4-O-methyl-d-glucuronic acid on every four xylose residues. Immunomodulatory activity assays in vitro showed that CTPB1 may induce the proliferation of mouse splenocytes. It was suggested that CTPB1 could be a potential immunostimulant used in the food industry.

The seed of Cassia obtusifolia is a food or herbal medicine used for improving eyesight, treating constipation and other disorders, and polysaccharides have been implicated in these pharmacological activities. The endosperm of the seeds, Cassia gum, is a commercial thickening or gelling agent, composed mainly of galactomannans. However, the whole seeds of C. obtusifolia, rather than the endosperm, are used in folk medicine or food, which might contain more complex constituents of polysaccharides. In this study, the whole seeds of C. obtusifolia were extracted with boiling water, and from the water extract, three homogeneous fractions were isolated, designated CFAA-1, CFAA-3, and CFBB2, respectively, after treatment with Fehling solution followed by anion-exchange and gel permeation chromatography. Using chemical and spectroscopic methods, CFAA-1, and CFAA-3 were elucidated to be both branched galactomannans with different molecular weights, consisting of 1,4-linked β-d-mannopyranosyl backbone with single-unit α-d-galactopyranosyl branches attached to O-6 of mannose, while CFBB2 was shown to be a linear (1 → 4)-α-polygalacturonic acid.Highlights► Three polysaccharides were isolated from whole seeds of Cassia obtusifolia. ► They were fractionated by Fehling solution complexing and chromatographic methods. ► CFAA-1 and CFAA-3 were elucidated to be both typical branched galactomannans. ► CFBB2 was shown to be a linear (1 → 4)-α-polygalacturonic acid.

A novel firefly luciferase inhibitor (3a) with a pyrrolo[2,3-d]pyrimidine core was identified in a cell-based NF-κB luciferase reporter gene assay. It potently inhibited the firefly luciferase derived from Photinus pyralis with an IC50 value of 0.36 ± 0.05 μM. Kinetic analysis of 3a inhibition showed that it is predominantly competitive with respect to d-luciferin and uncompetitive with respect to ATP. Therefore, several pyrrolo[2,3-d]pyrimidine analogues were prepared to further investigate the structure-activity relationship (SAR) for luciferase inhibition. The most potent inhibitor of this series was 4c, which showed an IC50 value of 0.06 ± 0.01 μM. In addition, molecular docking studies suggested that both 3a and 4c could be accommodated in the d-luciferin binding pocket, which is expected for a predominantly competitive inhibitor with respect to d-luciferin.

Ganoderma lucidum is an edible and medicinal mushroom used widely in East Asia. In recent years, its spores have been used as a supplement in combination with other forms of antitumor therapies. The cell wall of Ganoderma lucidum spores contains a high amount of polysaccharides. In this study, a neutral polysaccharide, GLSA50-1B, was isolated from sporoderm-broken spores of Ganoderma lucidum, by hot-water extraction, graded ethanol precipitation, anion-exchange chromatography, and gel permeation chromatography. Using sugar compositional analysis, methylation analysis, partial acid hydrolysis, acetolysis, and NMR and ESI-MS spectroscopy, GLSA50-1B was elucidated to be a novel β-d-glucan featured by a 1,6-linked β-d-Glcp backbone with different length of branches consisting of terminal and 1,4-linked Glcp residues, attached to O-4 of alternative Glc residues in the backbone.

Heparan sulfate proteoglycan (HSPG), such as glypican, plays a role as a co-receptor for growth factor to influence cells proliferation. However the mechanism is still vague. Micro-RNAs (miRNAs) regulate cell proliferation. Their capacity to direct the translation and stability of targeted transcripts can dramatically influence cellular physiological function. To explore how the function of glypican is regulated involved in cell proliferation, glypican-4 was chosen with a bioinformatics search identifying targeting seed sequences for miR-125a within the 3′-untranslated regions (3′UTR). Indeed, luciferase constructs containing the 3′UTR of glypican-4 demonstrated around 54 % less activity in miR-125a expressing cells relative to the controls. The expression of glypican-4 at both the transcript and protein level was down-regulated by transition trasfection of miR-125a in the human embryonic kidney cell line 293T (HEK293T). Although cell proliferation of HEK293T was not influenced by the silence of glypican-4, DNA synthesis in response to FGF2 in the cells was attenuated by knockdown of glypican-4 using siRNA technique. Further study showed that phosphorylation of ERK1/2 and AKT was suppressed by overexpressing miR-125a, whereas the suppressed MAPK and AKT signaling could be recovered by anti-miR-125a treatment. Both DNA synthesis and cell proliferation were impaired by the inhibitor of ERK1/2 signaling. MTT assay demonstrated that the cell proliferation was impaired by miR-125a overexpression, however, rescued by anti-miR-125a in HEK293T cells. These results disclosed new function of miR-125a by targeting gene glypican-4 in cell growth process and illustrated the feasibility of using miRNAs as a therapeutic strategy to suppress cells proliferation.

WGEW, an α(1-4) linked glucan with an α(1-4) linked branch attached to C-6, was isolated from the rhizoma of Gastrodia elata Bl. WSS25, a sulfated derivative of WGEW, was reported to inhibit angiogenesis by disrupting BMP2/Smad/Id1 signaling pathway. However, the structure-activity relationship (SAR) for WSS25 is not known. To study the SAR, seven sulfated saccharides derived from WGEW degradation products, six sulfated polysaccharides with varying degrees of substitution, and four aminopropylated, carboxymethylated, phosphorylated, and acetylated derivatives of WGEW were prepared. A sulfated, unbranched product of polysaccharide was also obtained. The structural features of these derivatives were characterized by infrared spectroscopy and nuclear magnetic resonance spectroscopy. An HMEC-1 cell tube formation assay was employed to measure the antiangiogenic effect of the derivatives. The results indicated that only sulfated polysaccharides with molecular weights of more than 41,000 Da could inhibit HMEC-1 cell tube formation. The inhibition effect was dependent on the presence of a sulfate group, since the tube formation was not blocked by aminopropylated, carboxymethylated, phosphorylated, or acetylated WGEW. A higher degree of sulfate substitution on the polysaccharide led to a stronger inhibitory effect, and the degree of sulfate substitution between 0.173 and 0.194 was found to be optimal. Interestingly, the WGEW side chain was not required for anti-tube formation activity. All these preliminary results may provide a clue for further modification of the core structure of WSS25 to discover polysaccharide derivatives as novel anti-angiogenic inhibitors.

A soluble homogeneous β-glucan, GFPBW1, with a molecular mass of 300 kDa was purified from the fraction of the fruit bodies of Grifola frondosa extracted with 5 % NaOH. Using various methods, such as infrared spectroscopy, NMR, methylation and monosaccharide composition analysis, its structure was determined to be a β-D-(1-3)-linked glucan backbone with a single β-D-(1-6)-linked glucopyranosyl residue branched at C-6 on every third residue. It induced TNF-α and IL-6 production and the activation of Syk and NF-κB signaling in resident peritoneal macrophages from ICR mice, which could be significantly inhibited by the blocking reagent laminarin. A competitive phagocytosis assay with FITC-zymosan indicated that GFPBW1 could bind to DC-associated C-type lectin 1 (Dectin-1). The TNF-α secretion and activation of Syk/NF-κB signaling triggered by GFPBW1 were enhanced in RAW264.7 cells overexpressing wild but not mutant (Δ38 and Y15S) Dectin-1. Furthermore, GFPBW1 potentiated the Concanavalin A-induced proliferative response of splenocytes and inhibited Sarcoma-180 growth allografted in ICR mice but not in immunodeficient BALB/c nu/nu mice. These results suggested that the antitumor activity of GFPBW1 was partially associated with the activation of macrophages via the Dectin-1/Syk/NF-κB signaling pathway. This molecule could be a promising biological response modifier with clear application for antitumor therapies.

To discover anti-pancreatic cancer cells growth inhibitor, a new water-soluble glucan, WTMA, was isolated from the rhizome of Gastrodia elata Bl. The mean molecular weight of the polysaccharide was estimated to be 7.0 × 105 Da by high performance gel permeation chromatography (HPGPC). The structure of WTMA was characterized by GC, GC–MS, NMR and MALDI-TOF. WTMA was deduced as an α-(1 → 4)-glucan with α-(1 → 4) linked branches attached to O-6 at branch points. Bioactivity tests in vitro indicated that WTMA and its partial acid hydrolysis-derived mixture product, WTMA-AD-O, could significantly inhibit the proliferation of pancreatic cancer cells. However, WTMA-DE, the degraded polysaccharide without branch chain by isoamylase, did not inhibit the proliferation of pancreatic cancer cells. Thus side chain on WTMA was deduced to be required for the anti-cancer cell activity. Further study demonstrated that WTMA and all of its hydrolysis products had no inhibition effect on liver LO2 cells.Highlights• The structure of 1,4-linked glucan with branch from Gastrodia elata was elucidated. • Glucan named WTMA could inhibit the proliferation of pancreatic cancer cells. • Hydrolysis-derived products from the glucan show more potent inhibition effect. • All the saccharides with anti-cancer cell growth bioactivity are with low toxicity. • O-6 attached branch of the glucan is required for anti-cancer cell growth bioactivity.

A polysaccharide, PGA4-3b, with an average molecular weight of 8.9 kDa estimated by high-performance gel-permeation chromatography (HPGPC), was isolated from radix of Platycodon grandiflorum (Jacq.) A. DC. Using monosaccharide analysis, methylation analysis and NMR spectroscopy, PGA4-3b was elucidated to be a linear poly-(1→4)-α-d-galactopyranosyluronic acid that contains no methyl ester groups. Partial acid hydrolysis of PGA4-3b yielded a series of poly- or oligogalacturonic acids with different degrees of polymerization (DP), that is, 4-3bde, 4-3bde-O-1, 4-3bde-O-2, 4-3bde-O-3, and 4-3bde-O-4. Cell tube formation inhibition tests with human microvascular endothelial cells (HMEC) for antiangiogenesis analysis showed that 4-3bde-O-1 and 4-3bde-O-2, the fractions with higher molecular weights, could inhibit tube formation, while the native PGA4-3b and low molecular weight fraction 4-3bde-O-3 and 4-3bde-O4 are ineffective. Moreover, 4-3bde-O-2 with DP 5-10 impaired cell tube formation in a dose-dependent way, suggesting its potential to be developed as an anti-angiogenesis drug. This is the first time oligogalacturonic acids are reported to show an anti-angiogenesis effect.PGA4-3b, a polygalacturonic acid was isolated from Platycodon grandiflorum. The oligogalacturonans prepared by partial hydrolysis of PGA4-3b showed inhibition on cell tube formation.

A water-soluble polysaccharide, PGAW1, with an average molecular mass of 9.2 kDa determined by high performance gel permeation chromatography (HPGPC), was isolated from radix of Platycodon grandiflorum. Monosaccharide composition analysis indicated that PGAW1 contains Ara and Gal in the molar ratio of 1.42:1.0. Using methylation analysis, partial hydrolysis, endo-1,4-β-d-galactanase digestion, NMR and ESI-MS, PGAW1 was determined to possess a backbone consisting of 1,4- and 1,6-linked galactopyranosyl residues, with branches attached to O-3 of 1,6-linked galactose residues. By the chlorosulfonic acid-pyridine method we produced a sulfated derivative of PGAW1, Sul-w1, with a substitution degree of 1.52. The substitution was at O-6 on 1,4-linked Gal residues according to the 13C NMR spectra. Bioactivity test showed that Sul-w1 could inhibit tube formation by human microvascular endothelial cells (HMEC) in a dose-dependent manner.

A polysaccharide fraction, J6, was isolated from the hot-water extract of flowers of oleander Nerium indicum Mill., using ethanol precipitation, cetyltrimethylammonium bromide (CTAB) complexing, anion-exchange chromatography and gel permeation chromatography. J6 was found to contain l-rhamnose, l-arabinose, d-galactose, and d-galacturonic acid, in the ratio of 10.1:49.8:30.1:10.0. Its structure was investigated by methylation analysis, periodate oxidation, Smith degradation, partial acid hydrolysis, electrospray ionization mass spectrometry and NMR spectroscopic methods. It was found that J6 is an RG-I type polysaccharide, which contains a rhamnogalacturonan backbone, with various branches attached to O-4 of l-rhamnose. The branches probably involve (1 → 4)-β-d-galactan, branched l-arabino-(1 → 3)(1 → 6)-β-d-galactan, and (1 → 5)-α-l-arabinan. J6 stimulated NO production of macrophage RAW264.7 cells in a preliminary test.A pectic polysaccharide, J6, isolated from the hot-water extract of the flowers of Nerium indicum Mill., contains a rhamnogalacturonan I backbone, with various branches attached to O-4 of rhamnose. J6 stimulates production of nitric oxide in RAW264.7 macrophage cells.

To discover drug candidates with anti-angiogenesis activity for cancer therapeutics, three galactooligosaccharides (OJ1–OJ3) were prepared by acid hydrolysis of the polysaccharides from Nerium indicum Mill. Their structures were characterized using sugar analysis, methylation analysis, and both 1D and 2D NMR spectroscopy, complemented by mass spectrometry. They were hexasaccharide (OJ1), a pentasaccharide (OJ2), and an undecasaccharide (OJ3), which was a new linear galactan. Bioactivity testing in vitro showed that OJ2 and OJ3 significantly inhibited the HMEC-1 (human microvascular endothelial cell) cell tube formation.Three oligosaccharides (OJ1–OJ3) were obtained by acid degradation of crude polysaccharides from Nerium indicum Mill. The study of chemical and spectroscopic methods demonstrated that they were β-d-Galp-(1→[4)-β-d-Galp-(1→]44)-d-Galp (OJ1), β-d-Galp-(1→[4)-β-d-Galp-(1→]34)-d-Galp (OJ2) and another new oligosaccharide, β-d-Galp-(1→[4)-β-d-Galp-(1→]94)-d-Galp (OJ3). Angiogenesis test showed that OJ2 and OJ3 blocked cell tube formation of HMEC-1.

A water soluble glucan, PLB-2C, was isolated from the water extract of the root of Pueraria lobata (Willd) Ohwi using anion-exchange and gel permeation chromatography. Its structure was investigated by gas chromatography (GC), gas chromatography–mass spectrometry (GC–MS), infrared (IR) spectra, and nuclear magnetic resonance (NMR) spectroscopy of heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) techniques. The results indicated that PLB-2C was a linear glucan composed of (1 → 6)-α-d-Glcp. Chain conformation study showed that the polysaccharide took random coil compact conformation. In vitro cell viability assay by MTT method, its sulfated derivative PLB-2CS which was substituted at 2-O, 3-O, 4-O positions, at 0.1, 1, and 5 mg/ml, could attenuate PC12 cell damage significantly caused by hydrogen peroxide.

A water-soluble polysaccharide (CPS-0) was obtained from the root of Cudrania tricuspidata (Carr.) Bur., by hot water extraction (70°C), deproteination using enzymolysis and Sevag method, precipitation with ethanol, and fractionation through DEAE-Sephadex A-50 chromatography. The purity of CPS-0 was determined by HPLC and the structure was elucidated by monosaccharide composition analysis, methylation analysis, GC, GC-MS, NMR spectral (1H-NMR, 13C-NMR, HMQC), UV, IR, and elemental analysis. The CPS-0 was found to contain glucose residues only. The average repeating unit is a decasaccharide having a backbone consisting of 1,4-linked α-D-glucopyranosyl residues to which the side chain consisting of terminal and 1,4-linked α-D-glucopyranosyl residues was attached at position 6 of the branching residues.

The structures of two glucans, WGEW and AGEW, isolated from Gastrodia elata Bl. were elucidated using monosaccharide composition analysis by gas chromatography (GC), methylation analysis by gas chromatography–mass spectrometry (GC–MS) and nuclear magnetic resonance (NMR) spectroscopy. Their structures were deduced as an α-d-(1→4)-glucan with an α-(1→4) linked branch attached to O-6 branch points with different branch degrees. Their sulfate derivatives with distinct degrees of substitution (DS) were prepared. The substitution position was assigned to O-6 according to the 13C NMR spectra. All sulfated derivatives showed strong anti-dengue virus bioactivities. The structure–activity relationships (SAR) between the polysaccharides and their sulfated derivatives were also investigated. Results showed that the higher the DS is, the more potent the impact on the dengue virus infection would be.