Rapid and efficient purification of chrysophanol in Rheum Palmatum LINN by supercritical fluid extraction coupled with preparative liquid chromatography in tandem.

Chrysophanol has high pharmaceutical values. However, it was difficult to use the traditional extraction method to extract high-concentration chrysophanol. Therefore, the purpose of this study is to purify and separate chrysophanol in traditional herb, Rheum Palmatum LINN, by using supercritical fluid extraction (SFE) and preparative high-performance liquid chromatography (P-HPLC) for rapid and large-scale isolation. The method is efficient for selective extraction of chrysophanol from the herbs, which have complex compositions. The extraction efficiency of chrysophanol with SFE is 25 × higher than that of boiled water extraction under the same extraction time. The optimal conditions for SFE were 210 atm and 85 °C for 30 min; for P-HPLC, a C18 column was used with a gradient elution of methanol and 1% acetic acid at a flow rate of 10 mL/min. According to (1)H NMR and LC-MS analyses, the purity of the isolated chrysophanol was as high as 99%. The recovery for chrysophanol in Rheum after SPE/PHPLC processing was in the range of 88-91.5%. Compared with other extraction and purification methods, the sequential system (SFE/P-HPLC) achieved the highest amount of extracted chrysophanol from Rheum Palmatum LINN (0.38 mg/g) and the shortest run time (3h). Hence, this rapid and environmentally friendly method can separate compounds based on polarity with high efficiencies and, coupled with P-HPLC, it may be applicable in the large-scale production of foods and medicines in the future.

Branched α-(1,4) glucans from Lentinula edodes (L10) in combination with radiation enhance cytotoxic effect on human lung adenocarcinoma through the Toll-like receptor 4 mediated induction of THP-1 differentiation/activation.

This work investigated the role of structure in the binding of polysaccharides from 10 regionally different strains of Lentinula edodes to Toll-like receptor 4 (TLR-4) on monocytes (THP-1) and the potential effect of this interaction on tumor cell viability. Principal component analysis and multiple linear regression identified arabinose, glucose 1 → 4 linkage, and molecular weights about 2700 and 534 kDa as the significant determinant factors associated with TLR-4 binding activity. The branched α-(1,4)-glucan (L10) had the strongest ability to bind to TLR-4 and induce THP-1 cell differentiation. L10 induction of the THP-1 cell differentiation, superoxide production, and cytokine production followed the TLR-4/MyD88/IKK/NFκB pathway. Coculture of irradiated human lung adenocarcinoma A549 cells with L10-activated THP-1 cells resulted in significantly decreased percentage of viable A549 cells from 66 to 37% (p = 0.018), increased levels of superoxide, interleukin-8, and RANTES, and decreased levels of angiogenin and vascular endothelial growth factor. The results indicate that L10-activated monocytes have the potential to boost the antitumor immune response and antitumor activity of radiotherapy.

Glycosyl linkage characteristics and classifications of exo-polysaccharides of some regionally different strains of Lentinula edodes by amplified fragment length polymorphism assay and cluster analysis.

We report here the first combined amplified fragment length polymorphism (AFLP) analysis of genomic DNA fingerprinting data and cluster analysis of the exo-polysaccharide glycosyl linkage data of 10 regionally different strains of Lentinula edodes to compare their genetic and structural similarities and differences. In addition, the monosaccharide compositions, molecular weights, glycosyl structural linkages were investigated for the exo-polysaccharides extracted from these different phylogenetic groups of regionally different L. edodes. All exo-polysaccharides had similar molecular weight distribution between 1x10(4) and 3x10(6) Da and the monosaccharide composition analysis revealed the presence of heterogeneous materials containing glucose, mannose, xylose, galactose, fucose, rhamnose and arabinose in different ratios. Among these monosaccharides, the glucose contents are the highest for all but one strain, indicating that glucose probably is the building block of the backbones of these exo-polysaccharides. The AFLP assay data helped to classify the 10 L. edodes strains into three distinct genetic groups. Gas chromatographic and mass spectrometric (GC-MS) data revealed five different glycosyl linkage types for these exo-polysaccharides. Most of the exo-polysaccharide backbone structures contain (1-->4)-linked-D-glucopyranosyl and (1-->6)-linked-D-glucopyranosyl moieties. Arabinose 1-->4 linkages and mannose 1-->2 linkages also exist in all strains. The only differences among these linkages are their monosaccharide compositions leading to different degree of backbone and branch formations. Cluster analyses of the GC-MS data of the exo-polysaccharides of the 10 strains resulted in 10 dendrograms. However, four of the 10 dendrograms were identical and were obtained using the average, Ward and weighted linkage type method of Manhattan distance and using the Ward method of Euclidean distance. The results of cluster analyses were not very much different from that of the AFLP assay and allowed the comparison of genetic and structural similarities and differences.

Pressurized water extraction of polysaccharides as secondary metabolites from Lentinula edodes.

The suitability of pressurized water extraction (PWE) of crude polysaccharides as secondary metabolites from Lentinula edodes was investigated. A series of experiments were carried out to examine the effects of extraction times and pressures. The results indicated that the maximum recovery of polysaccharides was about 90% of the crude polysaccharides from mycelia pellets when the pressure was at 10.1 MPa for 70 min (28 degrees C). This was a drastic improvement over that of boiling water extraction (BWE) at 0.1 MPa for 40 min, which gave only 27.9% recovery. A nitroblue tetrazolium (NBT) reduction assay was used to examine the macrophage stimulating activities (MSA), and it was found that the PWE polysaccharides retained the MSA. The morphology of the macrophage cells treated by PWE polysaccharides was also examined and found to be similar to that of the positive control lipopolysaccharides treated. Finally, gel chromatographic and NMR experiments revealed that both PWE and BWE polysaccharides showed the presence of four similar molecular mass components and the alpha-(1-->4)-D-Glcp and beta-(1-->6)-D-Glcp linkage residues. The improved PWE efficiency is probably due to the possibility that under high pressure, the solid polysaccharide's hydrogen bonding is partially destroyed to increase structure elasticity and water solubility.

Use of statistical methods to find the polysaccharide structural characteristics and the relationships between monosaccharide composition ratio and macrophage stimulatory activity of regionally different strains of Lentinula edodes.

Multiple linear regression analysis was used to deduce the correlation between the monosaccharide composition ratios of 10 regionally different strains of Lentinula edodes and their in vitro macrophage stimulatory activities. Arabinose, xylose, mannose and galactose were identified as the monosaccharides that could be related to macrophage stimulatory activities. Additional principal component analysis and factor analysis methods were used to treat the same monosaccharide composition ratio data and the compositions of arabinose, xylose, mannose and galactose were found to be important. Interestingly, glucose, although presented in large compositions in all strains presumably forms the backbone of the polysaccharide structures, is not selected as the determinant factor for either structural characteristics or that of the in vitro macrophage stimulatory activities.