Chemical components and molecular mass of six polysaccharides isolated from the sclerotium of Poria cocos

Six polysaccharides were extracted sequentially from the fresh sclerotium of Poria cocos cultivated in China using 0.9% NaCl (PCS1), hot water (PCS2), 0.5M NaOH (PCS3-I and PCS3-II), and 88% formic acid (PCS4-I and PCS4-II). Their chemical and physical characteristics were determined using infrared spectroscopy (IR), gas chromatography (GC), GC-MS methylation analysis, 13C NMR spectroscopy, elementary analysis (EA), protein analysis, size exclusion chromatography combined with laser light scattering (SEC-LLS), light scattering (LS), and viscometry. The results indicated that the polysaccharides PCS1, PCS2, and PCS3-I were heteropolysaccharides containing D-glucose, D-galactose, D-mannose, D-fucose, and D-xylose; the predominant monosaccharide was D-glucose except for PCS1 where it was D-galactose. PCS3-II, the main component of the sclerotium of P. cocos, was a linear (1-->3)-beta-D-glucan of high purity. PCS4-I consisted of (1-->3)-beta-D-glucan with some beta-(1-->6) linked branches. PCS4-II was mainly composed of (1-->3)-beta-D-glucan containing some glucose branches. The M(w) values of the six polysaccharides PCS1, PCS2, PCS3-I, PCS4-I in 0.2M NaCl aqueous solution, PCS3-II, and PCS4-II in dimethyl sulfoxide (Me(2)SO) were determined to be 11.6 x 10(4), 20.8 x 10(4), 17.1 x 10(4), 9.1 x 10(4), 12.3 x 10(4), and 21.1 x 10(4), respectively. The six polysaccharides in aqueous solution or Me(2)SO exist as flexible chains.     

Antitumor activities of heteropolysaccharides of Poria cocos mycelia from different strains and culture media

Ten water-soluble heteropolysaccharide fractions were isolated from Poria cocos mycelia cultured from one wild and one cultivated strain in two identical culture media differing only in one component: either corn steep liquor or bran extract. The chemical compositions, including monosaccharide profile, protein content, and molecular mass M(w) of the mycelial polysaccharides were determined. Both the in vitro and in vivo antitumor activities of the heteropolysaccharides were evaluated and compared. The heteropolysaccharides from Poria cocos mycelia cultured with the wild strain in a medium containing corn steep liquor exhibited the highest antitumor activities against Sarcoma 180 in vivo.     

Correlation of structure to antitumor activities of five derivatives of a beta-glucan from Poria cocos sclerotium

A water-insoluble (1-->3)-beta-D-glucan isolated from fresh sclerotium of Poria cocos was, respectively, sulfated, carboxymethylated, methylated, hydroxyethylated, and hydroxypropylated, to afford five water-soluble derivatives. Their weight-average molecular masses (Mw) and intrinsic viscosities ([eta]) were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in phosphate buffer solution (PBS) at 37 degrees C. The antitumor activities, against Sarcoma 180 tumor cell (S-180) and gastric carcinoma cell strain (MKN-45 and SGC-7901) of the native beta-glucan and the five derivatives, were tested in vitro and in vivo. The Mw values of the five derivatives in PBS were determined to be 3.8 x 10(4), 18.9 x 10(4), 16.0 x 10(4), 76.8 x 10(4), and 224.3 x 10(4), respectively. The high Mw values of the hydroxyethylated and hydroxypropylated derivatives in aqueous solution resulted from aggregation, and their true Mw values obtained in dimethyl sulfoxide were 20.1 x 10(4) and 19.1 x 10(4). The sulfated and carboxymethylated derivatives having DS of 1.0-1.3 show good water solubility, and exist as relatively expanded chains in aqueous solution. Interestingly, the native beta-glucan did not show antitumor activity, whereas the sulfated and carboxymethylated derivatives exhibit significant antitumor activities against S-180 and gastric carcinoma tumor cells. This work showed that good water solubility, relatively high chain stiffness, and moderate molecular mass of the derivatives in aqueous solution contribute beneficial to enhancement of antitumor activity.     

发酵茯苓菌丝体和天然茯苓多糖的研究

本文对发酵茯苓菌丝体和天然茯苓中多糖的提取分离进行了研究,并分别测定了二者总多糖的提取率及总糖的平均含量。发酵茯苓菌丝体和天然茯苓中总多糖的提取率分别为24.47%和82.93%;发酵茯苓菌丝体和天然茯苓总糖的平均含量分别为45.17%和87.24%。     

Solution properties of (1-->3)-alpha-D-glucan and its sulfated derivative from Poria cocos mycelia via fermentation tank

From Poria cocos mycelia yielded via a pilot scale facility-fermentation tank, a water-insoluble (1-->3)-alpha-D-glucan coded as Pi-PCM3-I was isolated by extraction with 0.5 M NaOH/0.01 M NaBH(4) aqueous solution. Nine fractions from F1 to F9 with a weight-average molecular mass (M(w)) range from 7.75 x 10(4) to 57.3 x 10(4) were prepared from the Pi-PCM3-I sample by a nonsolvent addition method. The fractions were reacted with chlorosulfonic acid-pyridine complex to product water-soluble sulfated derivatives coded as S1 to S8 with M(w) from 2.36 x 10(4) to 14.5 x 10(4) and degree of substitution (DS) of 0.86-1.38. M(w), z-average radius of gyration (s(2) (z) (1/2)), the second virial coefficient (A(2)), and the intrinsic viscosity ([eta]) of the native and sulfated Pi-PCM3-I were measured by laser light scattering (LLS), size-exclusion chromatography combined with LLS (SEC-LLS), and viscometry at 25 degrees C. The Mark-Houwink equations for Pi-PCM3-I in 0.25 M LiCl/dimethylsulfoxide (DMSO) (Me(2)SO) and for its sulfated derivative in 0.15 M NaCl aqueous solution at 25 degrees C were established to be [eta] = 1.33 x 10(-2) M(w) (0.75+/-0.01) (mL g(-1)) and [eta] = 1.46 x 10(-4) M(w) (1.13+/-0.01) (mL g(-1)), respectively. On the basis of theories for a wormlike cylinder model, the conformational parameters of the native and sulfated Pi-PCM3-I were calculated to be 760 +/- 50 and 1060 +/- 30 nm(-1) for the molar mass per unit contour length (M(L)), 6.3 +/- 0.5 and 13.1 +/- 1 nm for the persistence length (q), and 14.9 +/- 0.2 and 31.8 +/- 1 for the characteristic ratio (C( proportional, variant)), respectively. The results revealed that Pi-PCM3-I existed as an extended flexible chain in 0.25 M LiCl/Me(2)SO, and its sulfated derivative existed as a semistiff chain in 0.15 M NaCl aqueous solution. Furthermore, Pi-PCM3-I possessed similar structure and molecular parameters to wc-PCM3-I from a rotary shaker; this suggests promising industrialization of Poria cocos polysaccharides.     

Bioactive polysaccharides from the stems of the Thai medicinal plant Acanthus ebracteatus: their chemical and physical features

Crude water-soluble polysaccharides were isolated from Acanthus ebracteatus by hot water extraction followed by ethanol precipitation after pre-treatment with 80% ethanol. The crude polysaccharides were separated into neutral and acidic polysaccharides by anion-exchange chromatography. The neutral polysaccharide (A1001) was rich in galactose, 3-O-methylgalactose and arabinose, whereas the acidic polysaccharide (A1002) consisted mainly of galacturonic acid along with rhamnose, arabinose and galactose as minor components indicating a pectin-type polysaccharide with rhamnogalacturonan type I (RG-1) backbone. 3-O-Methylgalactose is also present in the acidic fraction. Both neutral and acidic fractions showed potent effects on the complement system using pectic polysaccharide PM II from Plantago major as a positive control. A small amount of 3-O-methylgalactose present in the pectin seemed to be of importance for activity enhancement in addition to the amount of neutral sugar side chains attached to RG-1. The relationship between chemical structure and effect on the complement system of the isolated polysaccharides is considered in the light of these data. The presence of the rare monosaccharide 3-O-methylgalactose may indicate that this can be used as a chemotaxonomic marker. The traditional way of using this plant as a medical remedy appears to have a scientific basis.     

Isolation and antioxidant activities of polysaccharides extracted from the shoots of Phyllostachys edulis (Carr.)

Polysaccharides extracted from Phyllostachys edulis (Carr.) are a group of hetero polysaccharides, and their antioxidant activities were investigated employing various established in vitro systems. Available data obtained with in vitro models suggested that among the three samples, B1 (extraction with water) showed significant inhibitory effects on superoxide radical and hydroxyl radical; its reducing power was also the strongest among the three samples. These results clearly establish the possibility that polysaccharides extracted from P. edulis could be effectively employed as ingredient in health or functional food, to alleviate oxidative stress. However, comprehensive studies need to be conducted in experimental animal models.     

Conformation of the exopolysaccharide of Burkholderia cepacia predicted with molecular mechanics (MM3) using genetic algorithm search

We present a computational conformational analysis of the exopolysaccharide of Burkholderia cepacia, which is believed to play a role in colonization and persistence of B. cepacia in the lungs of cystic fibrosis patients. The repeating unit of the exopolysaccharide is a heptasaccharide with three branches, which cause significant steric restraints. Conformational searches using glygal, an in-house developed software using genetic algorithm search methods, were performed on fragments as well as on the complete repeating unit with wrap-over residues. The force field used for the calculations was MM3(96). The search showed four favored conformations for an isolated repeating unit. However, for a sequence of several repeating units, the calculations indicate a single, well-defined linear conformation.     

The conformations of the O-specific polysaccharides of Shigella dysenteriae type 4 and Escherichia coli O159 studied with molecular mechanics (MM3) filtered systematic search

The branched O-antigens of Escherichia coli O159 and Shigella dysenteriae type 4 are structurally related and are known to show cross-reactivity with antibodies. In the present study, conformational analyses were performed on these two O-antigens using molecular mechanics MM3(96) with filtered systematic search. The results show very strong steric restrictions for the trisaccharide at the branch point of the E. coli O159 antigen, especially for the β-d-GlcNAc-(1 → 3)-β-d-GlcNAc linkage of the main chain. For the type 4 O-antigen the calculations show essentially a single conformation with respect to the α-d-GlcNAc-(1 → 3)-α-d-GlcNAc linkage of the main chain and three different favoured conformations for the fucose branch. Consecutive repeating units of the S. dysenteriae type 4 and E. coli O159 O-antigens form linear extended chains with significant flexibility between the branches. Comparative calculations carried out with the SWEET server indicate that our method of filtered systematic search is a superior method in the case of branched, constrained oligosaccharides. Based on the results of the MM3 calculations, we propose that the common epitope explaining the cross-reactivity comprises the fucose branch, the downstream GlcNAc and part of the uronic acid.     

Structural elucidation of fungal polysaccharides isolated from the cell wall of Plectosphaerella cucumerina and Verticillium spp

The structure of acidic fungal polysaccharides isolated from the cell wall of Plectosphaerella cucumerina, Verticillium dahliae, and V. albo-atrum has been investigated by chemical analysis, methylation analysis, and 1D and 2D 1H and 13C NMR spectroscopy. The polysaccharides have an idealized repeating block of the type: [carbohydrates: see text] linked to a small mannan core (<15%), where n=13, m=13, p=5, and q=8 for P. cucumerina, and n=16, m=16, p=6, and q <1 for both Verticillium species.     

Immunopontentiating and antitumor activities of the purified polysaccharides from Phellodendron chinese SCHNEID

The polysaccharide fractions were isolated and purified from Phellodendron chinese SCHNEID, and antitumor activities were examined at dosages of 2, 5 and 10 mg/100 g. F-7 and F-8 showed the highest tumor inhibitory activities (inhibition ratio 96.4 and 98.2% in 2 mg/100 g), and in dose of 5 mg/100 g, the inhibitory ratios were 95.3 and 97.5%, respectively. Furthermore, 10 mg/100 g of intraperitoneal (i.p.) injection gave 97.3 and 98.7% of inhibition. In oral administration, the inhibitory activities were not markedly observed, indicating that the polysaccharides are directly acting to immune system. Also the polysaccharides increased the number of circulating blood leukocytes and total peritoneal exudate cells. Although implantation of tumor cells greatly decreased the productivity of antibody (antibody-mediated) and T lymphocyte reactivity (delayed-type) as 6.3 from 9.3 and 5.9 from 7.7, represented by the increase of footpad thickness, respectively. The polysaccharides elevated the reactivity of T lymphocyte in tumor-bearing mice, which were rapidly recovered by discontinuance of sample treatments. Especially, F-2, F-5, F-7 and F-8 remarkably recovered the decreased sensitivity. When the effects on thymidylate synthase (TS) and thymidine kinase (TK) activities were determined, TS activities in the F-2 and F-7-treated mice were markedly suppressed to 73.7% and 79.5% of that in the control (p < 0.01), while there was little difference in TK activity with a slight decrease in F-2 only. However, in i.p. injection, TS activities in the F-2, F-5, F-7 and F-8-treated mice were markedly suppressed to 83% to 85% of that in the control (p < 0.01). Furthermore, there were also significant differences in TK activities in F-2, F-5, F-7 and F-8-treated mice (p < 0.05). These results clearly indicated that the i.p. injection is much effective to suppress tumor growth than oral administration.     

Fed-batch fermentation of Tuber melanosporum for the hyperproduction of mycelia and bioactive Tuber polysaccharides

For the first time, a fed-batch fermentation process of Tuber melanosporum was developed for the efficient production of bioactive mycelia and Tuber polysaccharides. Each 1.67 g/L of peptone and 8.33 g/L of yeast extract were added on day 3, 6, and 9, respectively, and sucrose was fed to maintain its concentration around 35–5 g/L when its residual level decreased to 10–5 g/L. Then, the maximal biomass, the production of extracellular polysaccharides (EPS) and intracellular polysaccharides (IPS) reached 53.72 ± 2.57 g DW/L, 7.09 ± 0.62 and 4.43 ± 0.21 g/L, respectively. Compared with the batch culture conducted in the enriched medium, the biomass, the production of EPS and IPS were enhanced by 55.8%, 222.3% and 103.2%, respectively. Not only the cell density but also the production of EPS and IPS were the highest ever reported in truffle fermentation, and the biomass was also the highest as ever reported in mushroom fermentation.     

Antioxidant activities of polysaccharides from Lentinus edodes and their significance for disease prevention

The crude polysaccharide (LEP) was extracted by hot water from the fruiting bodies of Lentinus edodes, and further purified by DEAE-cellulose and Sepharose CL-6B chromatography, giving three polysaccharide fractions coded as LEPA1, LEPB1 and LEPC1. In this study, their chemical and physical characteristics of polysaccharide fractions and antioxidant capacities, including scavenging activity against hydroxyl radicals, superoxide radicals and Fe²⁺-chelating ability, were valuated. The results showed that LEPC1 exhibited significantly antioxidant activity at a concentration-dependent manner. Therefore these results indicated that the water-extractable polysaccharide fraction was a potent antioxidant and could be developed to be new health medicine for fighting against various human diseases.     

Carboxymethylation of polysaccharides from Auricularia auricula and their antioxidant activities in vitro

The chemical carboxymethylated polysaccharide (CMAAP), which is derived from the poorly water-soluble, crude polysaccharide from Auricularia auricula (AAP), was prepared. The degree of substitution of CMAAP was 0.857, and its solubility in water reached 0.6 mg/mL, much higher than that of AAP (0.1 mg/mL). One fraction, CMAAP22, was purified. Chemical analysis indicated that CMAAP22 was composed of mannose and glucose in a ratio of 1.06:1 with a molecular weight of 3.4 × 10⁶ Da. IR spectra and ¹³C NMR spectra indicated that the C2, C4 and C6 positions were partially substituted by -CH₂COOH. AFM revealed that CMAAP22 was spherical particle structure with a diameter of 80-300 nm. Moreover, the antioxidant activity of CMAAP22 was improved, nearly twice as much as that of the AAP, especially in the scavenging of DPPH and ABTS+.     

Antioxidant and immunological activities of water-soluble polysaccharides from Aconitum kusnezoffii Reichb

Aconitum kusnezoffii Reichb., one of the earliest recorded toxic species of genus Aconitum, has been used as traditional Chinese medicine and medicinal diet over the last 2500 years to treat heart failure congestion, neuralgia, rheumatism and gout, etc. In the present paper, four water-soluble polysaccharide fractions isolated from the tubers of A. kusnezoffii Reichb. were studied the antioxidant and immunological activities for the first time. In vitro antioxidant assays indicated that fraction WKCP-A had noticeable scavenging activities on DPPH radical, hydroxyl radical, superoxide anion, H₂O₂ and self-oxidation of 1,2,3-phentriol, ferrous ion-chelating ability and reducing power. Moreover, the in vivo immunological assay exhibited that fractions WKCP-A and WKHP could more significantly enhance splenic lymphocyte proliferation and macrophage phagocytosis than other fractions. Therefore, the water-soluble polysaccharides from A. kusnezoffii Reichb., especially WKCP-A, have the potential to be explored as novel natural antioxidants and immunostimulating agents for using in functional foods or medicine.     

Molecular mass and antitumor activities of sulfated derivatives of α-glucan from Poria cocos mycelia

Two kinds of water-insoluble (1 → 3)-α-d-glucan samples, ab-PCM3-I and ac-PCM3-I, isolated from different Poria cocos mycelia were sulfated, to produce two series of water-soluble derivatives ab-PCM3-I-S1–S5 and ac-PCM3-I-S1–S5, respectively. The derivatives having different weight-average molecular mass (M_w) were produced by changing reaction temperature and time as well as molar ratios between chlorosulfonic acid and number of hydroxyl groups in the glucan. The degrees of substitution (DS) of the sulfated derivatives were analyzed by elemental analysis (EA) to be 0.39–0.67 for ab-PCM3-I-S and 0.73–0.96 for ac-PCM3-I-S, respectively. The M_w and the intrinsic viscosity ([η]) of the samples ab-PCM3-I-S and the ac-PCM3-I-S were measured by size exclusion chromatography combined with laser light scattering (SEC–LLS) and viscometry in phosphate buffer solution (PBS) at 37 °C. The results indicated that their M_w ranged from 2.0 to 11.3 × 10⁴ for the samples ab-PCM3-I-S, and 4.7 to 40.0 × 10⁴ for the samples ac-PCM3-I-S. Moreover, the antitumor activities of the sulfated derivatives ab-PCM3-I-S and ac-PCM3-I-S against Sarcoma 180 tumor cell tested both in vitro and in vivo are significantly higher than those of the native α-d-glucans. Therefore, a moderate range of molecular mass from 2.0 × 10⁴ to 40.0 × 10⁴, relatively high chain stiffness and good water solubility of the sulfated derivatives are beneficial to the enhancement of their antitumor activities.     

Triterpenes from the fungus Poria cocos and their inhibitory activity on nitric oxide production in mouse macrophages via blockade of activating protein-1 pathway

Two new triterpenes, 29-hydroxydehydrotumulosic acid (1) and 29-hydroxydehydropachymic acid (2), together with six known compounds, dehydropachymic acid (3), dehydrotumulosic acid (4), 29-hydroxypolyporenic acid C (5), polyporenic acid C (6), tumulosic acid (7), and pachymic acid (8), were isolated from the dried sclerotia of Poria cocos. In the in vitro bioassays, these isolated compounds reduced, in a dose-dependent manner, nitric oxide (NO) production from lipopolysaccharide (LPS)-induced RAW 264.7 cells, with compounds 5 and 6, the IC(50) values of which were 16.8±2.7 and 18.2±3.3 μM, respectively, exhibiting the greatest inhibition activity. Further Western blot analysis conducted on cells pre-treated with compounds 5 and 6, and luciferase assays on activator protein 1-dependent gene expression revealed that the inhibited NO release was attributed to the reduced expression of iNOs (=inducible NO synthase) enzymes, which might be regulated via the blockade of activator protein-1 signaling pathway.     

Experimental analysis of the oil addition effect on mycelia and polysaccharide productions in Ganoderma lucidum submerged culture

The effect of corn oil addition on mycelium growth and polysaccharide productions in the medicinal mushroom Ganoderma lucidum was studied. The results showed that when a level of 2% corn oil was added at the beginning of culture, the biomass and polysaccharide productions reached a maximum of 12.9 and 1.038 g/L, respectively, during 13-day cultivation. The pH variation along with morphology observation in culture provided an indirect inference to the promotional effect of oil addition. Moreover, a curve fitting analysis was carried out to assay the elevated effect on biomass and exopolysaccharide productions in oil added culture. The experimental data of substrates consumption and products formation in culture with oil addition were predicted through the fitting equations obtained in single carbon source culture. The numerical results further clarified the stimulatory effects of oil addition in G. lucidum culture.     

Phylogenetic diversity of Flavobacteria isolated from the North Sea on solid media

Flavobacteria are abundant in the North Sea, an epeiric sea on the continental shelf of Europe. However, this abundance has so far not been reflected by the number of strains in culture collections. In this study, Flavobacteria were isolated from pelagic and benthic samples, such as seawater, phytoplankton, sediment and its porewater, and from surfaces of animals and seaweeds on agar plates with a variety of carbon sources. Dilution cultivation with a new medium, incubation at low temperatures and with long incubation times, and colony screening by a Flavobacteria-Cytophagia-specific PCR detecting 16S rRNA gene sequences led to a collection of phylogenetically diverse strains. Two strains affiliated with Flammeovirgaceae and seven strains affiliated with Cyclobacteriaceae, whereas within the Flavobacteriaceae 20 isolated strains presumably represented seven novel candidate genera and 355 strains affiliated with 26 of 80 validly described marine Flavobacteriaceae genera, based on a genus boundary of 95.0% 16S rRNA gene sequence identity. The majority of strains (276) affiliated with 37 known species in 16 genera (based on a boundary of 98.7% 16S rRNA gene sequence identity), whereas 79 strains likely represented 42 novel species in 22 established Flavobacteriaceae genera. Pigmentation, iridescence, gliding motility, agar lysis, and flexirubin as a chemical marker supported the taxonomy at the species level. This study demonstrated the culturability on solid medium of phylogenetically diverse Flavobacteria originating from the North Sea.