Structural characterization of novel arabinoxylan and galactoarabinan from citron with potential antitumor and immunostimulatory activities.

This study aimed to extract polysaccharides from citron and analyze their structures and potential bioactivities. Two novel polysaccharides CM-1 and CM-2 were purified from citron by DEAE-Sepharose Fast Flow and Sephadex G-100 column chromatography. Monosaccharide composition, linkage and NMR data were used to infer their sugar chains composition. The anti-breast cancer cells and immunoregulatory activities of CM-1 and CM-2 were investigated. Results indicated that CM-1 (Mw = 21,520 Da), composed of arabinose, xylose, mannose and glucose in a molar ratio of 10.78:11.53:1.00:1.70, was arabinoxylan (AX) with (1 â†’ 4)-linked ß-d-Xylp skeleton monosubstituted with α-l-Araf units at O-3 position. While CM-2 (Mw = 22,303 Da), composed of arabinose, mannose, glucose and galactose in a molar ratio of 25.46:1.45:1.00:6.57, was galactoarabinan (GA) with (1 â†’ 5)-linked α-l-Araf backbone substituted by ß-d-Galp units at O-2 and/or O-3 positions. Both polysaccharides exhibited potential inhibiting cancer and immunostimulatory activities in vitro, especially CM-1. These results provide a basis for further research on citron polysaccharides.

Extraction, characterization of xylan from Azadirachta indica (neem) sawdust and production of antiproliferative xylooligosaccharides.

Xylan extracted from neem sawdust gave 22.5%, (w/w) yield. The extracted xylan was composed of xylose and glucuronic acid at a molar ratio of 8:1 and with a molecular mass, ~66 kDa. FTIR and NMR analyses indicated a backbone of xylan substituted with 4-O-methyl glucuronic acid at the O-2 position. FESEM analysis showed a highly porous and granular surface structure of xylan. A thermogravimetric study of xylan showed thermal denaturation at 271 °C. The hydrolysis of xylan by recombinant endo-ß-1,4-xylanase produced a mixture of xylooligosaccharides ranging from degree of polymerization 2-7. Xylooligosaccharides inhibited cell growth of human colorectal cancer (HT-29) cells but did not affect the mouse fibroblast cells confirming its biocompatibility. Western blotting, DNA laddering and flow cytometric analysis displayed inhibition of HT-29 cells by xylooligosaccharides. FLICA staining and mitochondrial membrane potential analyses confirmed the activation of the intrinsic pathway of apoptosis. The study amply indicated that the xylooligosaccharides produced from neem xylan could be potentially used as an antiproliferative agent.

Chemopreventive role of arabinoxylan rice bran, MGN-3/Biobran, on liver carcinogenesis in rats.

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and one of the most lethal. MGN-3/Biobran is a natural product derived from rice bran hemicelluloses and has been reported to possess a potent anticancer effect in a clinical study of patients with HCC. The current study examines the mechanisms by which Biobran protects against chemically induced hepatocarcinogenesis in rats. The chemical carcinogen used in this study is N-nitrosodiethylamine (NDEA) plus carbon tetrachloride (CCl4). Rats were treated with this carcinogen, and the animals were pretreated or posttreated with Biobran via intraperitoneal injections until the end of the experiment. Treatment with Biobran resulted in: 1) significant alleviation of liver preneoplastic lesions towards normal hepatocellular architecture in association with inhibition of collagen fiber deposition; 2) arrest of cancer cells in the sub-G1 phase of the cell cycle; 3) increased DNA fragmentation in cancer cells; 4) down-regulated expression of Bcl-2 and up-regulated expression of p53, Bax, and caspase-3; and 5) protection against carcinogen-induced suppression of IkappaB-alpha (IκB-α) mRNA expression and inhibition of nuclear factor kappa-B (NF-κB/p65) expression. Additionally, the effect of Biobran treatment was found to be more significant when supplemented prior to carcinogen-induced hepatocarcinogenesis as compared to posttreatment. We conclude that Biobran inhibits hepatocarcinogenesis in rats by mechanisms that include induction of apoptosis, inhibition of inflammation, and suppression of cancer cell proliferation. Biobran may be a promising chemopreventive and chemotherapeutic agent for liver carcinogenesis.

Preparation, structural characterization and bioactivity of 4-O-Methylglucuronoxylan from Artemisia sphaerocephala Krasch.

We obtained four soluble acid xylan fractions AGP-III-A, AGP-III-B, AGP-III-C and AGP-III-D from the insoluble Artemisia sphaerocephala Krasch gum (ASKG) polysaccharide by weak alkali treatment combined with H2O2-Vc oxidative degradation. Activity studies showed that the degradation components could reduce the cell viability of several cancer cell lines in a concentration-dependent manner, especially 4-O-Methylglucuronoxylan AGP-III-C with specific molecular weight and branching degree significantly reduced cancer cells viability and induced HepG2 apoptosis, also caused mitochondrial membrane dysfunction upregulated ROS levels, and induced G0/G1 arrest in HepG2 cells by cell cycle assay. Further, AGP-III-C mediates apoptosis in HepG2 cells by upregulating MAPK phosphorylation. The structure of AGP-III-C was characterized by uronic acid reduction, permethylation with GC-MS, and 2D-NMR analysis. The structure of AGP-III-C had a linear (1→4)-linked ß-Xylf residue backbone with one branched 4-O-Me-α-GlcAp attached to the main chain by a (1→2)-glycosidic bond at every two ß-(1→4)-Xylf units.

Structural and antioxidant studies of a new arabinoxylan from green stem Andrographis paniculata (Kalmegh).

A water soluble arabinoxylan (APPS), with a molecular weight ∼1.49 × 105 Da was isolated from the green stem of Andrographis paniculata. The APPS contained D-xylose, 2-methoxy D-xylose and L- arabinose in molar ratio of 3:1:1. The structure of the repeating unit in the polysaccharide was determined through several chemical (acid hydrolysis, Methylation analysis and periodate oxidation) and spectroscopic analysis (1D/2D NMR experiments) as: In vitro antioxidant assay, APPS was found to possess ferrous ion chelating activity (EC50 = 283 µg/ml), superoxide radical scavenging activity (EC50 = 470 µg/ml), and hydroxide radical scavenging activity (EC50 = 193 µg/ml). Thus, APPS could be used as a natural antioxidant agent for food and pharmaceutical industries.

Hydroxycinnamic acid bound arabinoxylans from millet brans-structural features and antioxidant activity.

Hydroxycinnamic acid bound arabinoxylans (HCA-AXs) were extracted from brans of five Indian millet varieties and response surface methodology was used to optimize the extraction conditions. The optimal condition to obtain highest yield of millet HCA-AXs was determined as follows: time 61min, temperature 66°C, ratio of solvent to sample 12ml/g. Linkage analysis indicated that hydroxycinnamic acid bound arabinoxylan from kodo millet (KM-HCA-AX) contained comparatively low branched arabinoxylan consisting of 14.6% mono-substituted, 1.2% di-substituted and 41.2% un-substituted Xylp residues. The HPLC analysis of millet HCA-AXs showed significant variation in the content of three major bound hydroxycinnamic acids (caffeic, p-coumaric and ferulic acid). The antioxidant activity of millet HCA-AXs were evaluated using three in vitro assay methods (DPPH, FRAP and ß-carotene linoleate emulsion assays) which suggested both phenolic acid composition and structural characteristics of arabinoxylans could be correlated to their antioxidant potential, the detailed structural analysis revealed that low substituted KM-HCA-AX exhibited relatively higher antioxidant activity compared to other medium and highly substituted HCA-AXs from finger (FM), proso (PM), barnyard (BM) and foxtail (FOXM) millet.

Influence of grain particle sizes on the structure of arabinoxylans from brewer's spent grain.

BSG was milled and sieved and six different grain fractions of different particle sizes (PS) were obtained: PS ≤ 63 µm (PSA); 63 µm < PS ≤ 90 µm (PSB); 90 µm250 µm (PSF). SEM images showed that for the highest particle sizes, cells were still intact while for the smallest particles disruption of cells occurred. The sugar analysis of the grains showed the constant presence of arabinoxylans (AX) and the maximum amount was observed in PSC. AX rich extracts were obtained for all grain fractions presenting different estimate degrees of polymerisation (DP) and degrees of branching (DB). With the decreasing of grain PS, smallest and more branched polymers were extracted; the smallest AX in PSC, and the more branched AX in PSA and PSB. Residual arabinogalactans (AG) were extracted from PSD to PSA. Starch was present in all extracts accounting for 8-10% of the composition of the extract. AX rich extracts with different DP and DB were obtained from different grain PS.

Evaluation of the prebiotic potential of arabinoxylans from brewer's spent grain.

Arabinoxylans (AX) consumption has been related to the treatment and prevention of cardiovascular diseases, type II diabetes, colorectal cancer and obesity. The beneficial health effects are conferred through gut microbiota modulation, and therefore, they have been proposed as potential slowly fermentable prebiotic candidates. As the mechanisms are not yet well understood, the prebiotic potential of AX from brewer's spent grain (BSG) has been investigated. Two types of AX from BSG (AX1 and AX2) of different length and branching averages were fermented with human faecal inocula and compared to fermented cultures containing a commercial prebiotic (fructooligosaccharide (FOS)) and cultures with no added carbohydrate (control). Results demonstrated that the AX were extensively metabolised after 48 h of fermentation. The pH decreased along fermentation and the lowest value was achieved in AX1 cultures. The production of short chain fatty acids (SCFA) was higher in AX cultures than in cultures containing FOS and controls, with AX1 presenting the highest concentrations. The stimulatory effect of beneficial bacteria was higher in AX cultures, and AX2 presented the highest positive effect. Prebiotic potential of AX from BSG was confirmed by the production of SCFA and the modulation of gut microbiota, especially by the high increase in bifidobacteria populations.

Structure and activities of a novel heteroxylan from Cassia obtusifolia seeds and its sulfated derivative.

COB1B1S2 was isolated from an alkaline extract of Cassia obtusifolia seeds, and purified by anion-exchange and gel permeation chromatography. It contains arabinose, xylose, and glucuronic acid, in the molar ratio of 5:81:14, with an apparent molecular weight estimated to be 70.4 kDa. Elucidated by using chemical and spectroscopic methods, COB1B1S2 was shown to have a backbone consisting of 1,4-linked ß-D-Xylp, with one single-unit terminal α-D-GlcpA or α-L-Araf substituted at O-2 for nearly every five 1,4-linked Xylp. COB1B1S2 is structurally different from typical glucuronoxylans by its absence of methylation at O-4 of GlcA. The native COB1B1S2 showed no significant inhibition on the tube formation of human microvascular endothelial cells (HMEC) and on the growth of liver and colon cancer cells. On the contrary, COB1B1S2-Sul, prepared as the sulfated derivative of COB1B1S2, exhibited a significant inhibition on tube formation of HMEC in a dose-dependent manner, and on the growth of Bel7402 liver cancer cells. These results indicated that the introduction of sulfate groups significantly enhanced the biological activity of glucuronoxylan.

Structural features of two heteroxylan polysaccharide fractions from wheat bran with anti-complementary and antioxidant activities.

Wheat bran is a rich source of bioactive substances ascribed to its arabinoxylan component. Two water-soluble arabinoxylans were sequentially extracted from wheat bran. WB1, released during enzymatic digestion of starch and protein, contained medium-branched arabinoxylan (A/X=0.88) consisting of 3-O-substituted (22%), di-substituted (19.8%) and 58% unsubstituted Xylp residues. It was slightly contaminated with (1→3,1→4)-ß-glucan and arabinogalactan, and free of protein. WB2 extracted with 0.5% NaOH contained ∼95% arabinoxylan (A/X=1.09). WB2 and two 5% NaOH-extracted arabinoxylans were rich in protein and phenolic compounds. All radical-scavenging assays indicated a relation with the protein and total phenolics contents. The protein-free WB1 displayed the highest hydroxyl radical scavenging effect indicating the distinct role of phenolic acids. The immunomodulatory activity of WB1 was somewhat lower, whereas, that of WB2 higher in comparison to the immunogenic polysaccharide PMII. The arabinoxylans have the potential as immuno-enhancing and antioxidant additives in functional foods.

A new arabinoxylan from green leaves of Litsea glutinosa (Lauraeae): structural and biological studies.

A water soluble new arabinoxylan, isolated through hot water extraction from the green leaves of Litsea glutinosa (Lauraceae) was found to contain xylose and arabinose in a molar ratio of nearly 1:3. On the basis of NMR ((1)H, (13)C, DQF-COSY, TOCSY, NOESY, ROESY, HMBC and DEPT-135), GLC and GLC-MS analyses, the backbone was established as (1→4)-α-D-xylopyranosyl residue, substituted at C-2 with one unit of two adjacently linked (1→3)-α-L-arabinofuranosyl residues and the other one was terminated by ß-L-arabinofuranosyl residue. The proposed repeating unit of the molecule was established as: [formula, see text] This molecule showed strong splenocyte, thymocyte, and macrophage activations. The optimum doses of the polysaccharide for splenocyte and thymocyte proliferation were observed at 25 µg/mL and 50 µg/mL, respectively. An enhanced production of NO was observed at 100 µg/mL of the polysaccharide.

Utilization of Agro-industrial Wastes for the Simultaneous Production of Amylase and Xylanase by Thermophilic Actinomycetes

Agro-industrial wastes such as sugarcane bagasse, wheat bran, rice bran, corn cob and wheat straw are cheapest and abundantly available natural carbon sources. The present study was aimed to production of amylase and xylanase simultaneously using agro-industrial waste as the sole carbon source. Seven thermophilic strains of actinomycete were isolated from the mushroom compost. Among of these, strain designated MSC702 having high potential to utilize agro-industrial wastes for the production of amylase and xylanase. Strain MSC702 was identified as novel species of Streptomyces through morphological characterization and 16S rRNA gene sequence. Enzyme production was determined using 1% (w/v) of various agro-industrial waste in production medium containing (g/100mL): K2HPO4(0.1), (NH4)2SO4(0.1), NaCl (0.1), MgSO4(0.1) at pH 7.0 after incubation of 48 h at 50°C. The amylase activity (373.89 IU/mL) and xylanase activity (30.15 IU/mL) was maximum in rice bran. The decreasing order of amylase and xylanase activity in different type of agro-industrial wastes were found rice bran (RB) > corn cob (CC) > wheat bran (WB) > wheat straw (WS) > sugarcane bagasse (SB) and rice bran (RB) > wheat bran (WB) > wheat straw (WS) > sugarcane bagasse (SB) > corn cob (CC), respectively. Mixed effect of different agro-industrial wastes was examined in different ratios. Enzyme yield of amylase and xylanase was ~1.3 and ~2.0 fold higher with RB: WB in 1:2 ratio.

Induction of apoptosis in MCF-7 cells by ß-1,3-xylooligosaccharides prepared from Caulerpa lentillifera.

ß-1,3-Xylan was prepared from the green alga, Caulerpa lentillifera, and hydrolyzed to oligosaccharides by a mild acid treatment. The average degree of polymerization was about 5. The oligosaccharides reduced the number of viable human breast cancer MCF-7 cells in a dose-dependent manner, and induced chromatin condensation and degradation of poly ADP-ribose polymerase, indicating that they induced apoptosis in MCF-7 cells.

A biodegradation study of forest biomass by Aspergillus niger F7: correlation between enzymatic activity, hydrolytic percentage and biodegradation index

Aspergillus niger F7 isolated from soil was found to be the potent producer of cellulase and xylanase. The residue of forest species Toona ciliata, Celtris australis, Cedrus deodara and Pinus roxburghii was selected as substrate for biodegradation study due to its easy availability and wide use in industry. It was subjected to alkali (sodium hydroxide) treatment for enhancing its degradation. Biodegradation of forest waste by hydrolytic enzymes (cellulase and xylanase) secreted by A. niger under solid state fermentation (SSF) was explored. SSF of pretreated forest biomass was found to be superior over untreated forest biomass. Highest extracellular enzyme activity of 2201±23.91 U/g by A. niger was shown in pretreated C. australis wood resulting in 6.72±0.20 percent hydrolysis and 6.99±0.23 biodegradation index (BI). The lowest BI of 1.40±0.08 was observed in untreated saw dust of C. deodara having the least enzyme activity of 238±1.36 U/g of dry matter. Biodegradation of forest biomass under SSF was increased many folds when moistening agent i.e. tap water had been replaced with modified basal salt media (BSM). In BSM mediated degradation of forest waste with A. niger, extracellular enzyme activity was increased up to 4089±67.11 U/g of dry matter in turn resulting in higher BI of 15.4±0.41 and percent hydrolysis of 19.38±0.81 in pretreated C. australis wood. A. niger exhibited higher enzyme activity on pretreated biomass when moistened with modified BSM in this study. Statistically a positive correlation has been drawn between these three factors i.e. enzyme activity, BI and percent hydrolysis of forest biomass thus proving their direct relationship with each other.

The hydrolysis of agro-industrial residues by holocellulose-degrading enzymes

Holocellulose structures from agro-industrial residues rely on main and side chain attacking enzymes with different specificities for complete hydrolysis. Combinations of crude enzymatic extracts from different fungal species, including Aspergillus terreus, Aspergillus oryzae, Aspergillus niger and Trichoderma longibrachiatum, were applied to sugar cane bagasse, banana stem and dirty cotton residue to investigate the hydrolysis of holocellulose structures. A. terreus and A. oryzae were the best producers of FPase and xylanase activities. A combination of A. terreus and A. oryzae extracts in a 50% proportion provided optimal hydrolysis of dirty cotton residue and banana stem. For the hydrolysis of sugar cane bagasse, the best results were obtained with samples only containing A. terreus crude extract.

Thermostability of xylanolytic enzymes produced by Lentinula edodes UFV70

Xylanolytic enzymes produced by Lentinula edodes UFV70, cultivated in eucalyptus sawdust/rice bran medium, were stable at 50, 60 and 65ºC for 21 hours, losing only 15-25% activity. Fungus incubation at 50ºC for 12 hours and at 65ºC for 24 hours increased the amount of xylose produced.

Hyperexpression of two Aspergillus niger xylanase genes in Escherichia coli and characterization of the gene products

The analysis of individual gene product should enable to clarify the role of a particular enzyme in a complex xylanase system of A. niger. The two genes encoding precursors of co-produced endo-1,4-¥â-D-xylanases, xynA1 and xynB, were isolated from Aspergillus niger SCTCC 400264 (SCTCC, China) by using RT-PCR technique and then successfully expressed in Escherichia coli BL21. The nucleotide sequences of the xynA1 and xynB genes revealed that they were only 52.5 percent homology to each other. Characterization of the recombinant enzymes revealed the different properties: the specific activity of recombinant XYNA1 was 16.58 U/mg compared to 1201.7 U/mg for recombinant XYNB; The optimum temperature and pH of the recombinant XYNA1 were 35 ¨¬C and 3.0, respectively, whereas the corresponding values for the recombinant XYNB were 55 ¨¬C and 5.0, respectively; The recombinant XYNB showed much more thermostability than recombinant XYNA1; The recombinant XYNB showed 94 percent of maximal activity after incubating in water for 60 min at 60 ¨¬C compared to no activity for recombinant XYNA1. Various metal ions had different effects on activity between the two recombinant xylanases.

One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

A purificação de uma etapa e caracterização de uma xilanase livre de celulase de uma linhagem recentemente isolada alcalofílicos e moderadamente termofílico de Bacillus subtilis ASH. Xilanase foi purificada à homogeneidade de 10,5 vezes, com ~ por cento de recuperação 43 através de cromatografia de troca iônica através de CM- Sephadex C -50. A enzima purificada revelou uma única banda no gel SDS-PAGE com uma massa molecular de 23 kDa. Ele mostrou um pH ótimo de 7,0 e manteve-se estável na faixa de pH 6,0-9,0 . A temperatura ótima para atividade da enzima foi 55 º C. A xilanase purificada não perder nenhuma atividade até 45 º C , no entanto, manteve 80 por cento e 51 por cento de sua atividade após pré-incubação a 55 º C e 60 º C , respectivamente. A enzima obedecido Michaelis- Menton cinética para xilano de madeira de bétula com aparente km 3,33 mg / ml e Vmax 100 UI / ml. A enzima foi fortemente inibida por Hg2 +, Cu2 + , enquanto reforçada por Co2 + e Mn2 +. A enzima purificada pode ser armazenado a 4 º C por seis semanas sem nenhuma perda de atividade catalítica. A purificação mais rápido e econômico da xilanase livre de celulase de B. subtilis ASH por um passo-a processo juntamente com a sua estabilidade sensível a alta temperatura e pH alcalino torna potencialmente eficazes para aplicações industriais.

Discovery and characterization of cadherin domains in Saccharophagus degradans 2-40.

Saccharophagus degradans strain 2-40 is a prominent member of newly discovered group of marine and estuarine bacteria that recycle complex polysaccharides. The S. degradans 2-40 genome codes for 15 extraordinary long polypeptides, ranging from 274 to 1,600 kDa. Five of these contain at least 52 cadherin (CA) and cadherin-like (CADG) domains, the types of which were reported to bind calcium ions and mediate protein/protein interactions in metazoan systems. In order to evaluate adhesive features of these domains, recombinant CA doublet domains (two neighboring domains) from CabC (Sde_3323) and recombinant CADG doublet domains from CabD (Sde_0798) were examined qualitatively and quantitatively for homophilic and heterophilic interactions. In addition, CA and CADG doublet domains were tested for adhesion to the surface of S. degradans 2-40. Results showed obvious homophilic and heterophilic, calcium ion-dependent interactions between CA and CADG doublet domains. Likewise, CA and CADG doublet domains adhered to the S. degradans 2-40 surface of cells that were grown on xylan from birch wood or pectin, respectively, as a sole carbon source. This research shows for the first time that bacterial cadherin homophilic and heterophilic interactions may be similar in their nature to cadherin domains from metazoan lineages. We hypothesize that S. degradans 2-40 cadherin and cadherin-like multiple domains contribute to protein-protein interactions that may mediate cell-cell contact in the marine environment.

Cereal non-cellulosic polysaccharides: structure and function relationship - an overview.

The non-cellulosic polysaccharides present in cereals (2-8%) are mostly arabinoxylans, (1 --> 3),(1 --> 4)-beta -glucans, pectins and arabinogalactans. Of these, the arabinoxylans are known to absorb large amounts of water and influence significantly the water balance, rheological properties of dough, and the retrogradation of starch and bread quality. (1 --> 3),(1 --> 4)-beta -glucans are known as biological response modifiers (BMS) as they are believed to modulate the immune response. Cereal Pectins and arabinogalactans form a very small amount and do not contribute substantially to the functionality of noncellulosic polysaccharides. Detailed structural investigations on cereal hetero xylans using modern techniques were initiated in the 1990s and still pose a challenge to carbohydrate chemists because of their structural complexity. Nutritionally, they are classified under "unavailable carbohydrates" (dietary fiber) along with lignin and cellulose and are known to have beneficial effects in alleviating disease symptoms such as diabetes, atherosclerosis, and colon cancer. In this review isolation, purification, characterization, structural elucidation, functional, and nutritional attributes of cereal heteroxylans are covered with particular emphasis on recently characterized finger millet arabinoxylans.