An oligosaccharide-marker approach to quantify specific polysaccharides in herbal formula by LC-qTOF-MS: Danggui Buxue Tang, a case study.

Polysaccharides have broad bioactivities and are major components of water decoction of herb formulae. However, the quality control of polysaccharides remains a challenge. Oligosaccharide-fragment approach has been considered in elucidating chemical structures of polysaccharides, but never been used for quantitation. Using reference chemicals and a real sample Danggui Buxue Tang (DBT) in this study, an oligosaccharide-marker approach was established to quantify specific polysaccharides. Firstly, linear relationships between parent polysaccharides and hydrolysis-produced daughter oligosaccharides were verified using reference polysaccharides. Then in case of DBT, two fluorescence-labeled oligosaccharides with high specificity to individual parent polysaccharides were selected as markers. They were easily isolated and identified. Their potential in quantification of parent polysaccharides were satisfactorily validated in terms of linearity (r≥0.99), repeatability (RSD ≤ 8.4 %), and spike recovery (≥80 %). This method could be a promising approach for quality assessment of polysaccharides in herbal formulae.

TLR-4 may mediate signaling pathways of Astragalus polysaccharide RAP induced cytokine expression of RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Polysaccharides of Radix Astragali (Astragalus membranaceus (Fisch) Bge.; Huangqi) are able to induce cytokine production of macrophages and are considered the main active ingredient for the immune-enhancing effect of this commonly used medicinal herb. AIM OF STUDY: To investigate the molecular mechanism of immunomodulating activities of a reported Astragalus polysaccharide, RAP, which is a hyperbranched heteroglycan with average molecular weight of 1334kDa. MATERIALS AND METHODS: The cytokine production of RAW264.7 cells were analyzed by using ELISA assays while cell viability was assessed by MTT method. Western blot analysis was used for determining protein contents of mitogen-activated protein kinases (MAPKs). In addition, the level of IL-6, iNOS, and TNF-α mRNA was determined by RT-PCR. RESULTS: It has been found that RAP itself did not have any cytotoxic effect on mouse mammary carcinoma 4T1 cells, but it significantly enhanced cytotoxicity of the supernatant of RAW264.7cells on 4T1 cells. Furthermore, RAP enhanced the production of NO and cytokines in RAW264.7 cells, and significantly up-regulated gene expressions of TNF-α, IL-6, iNOS. All these bioactivities were blocked by the inhibitor of TLR4 (Toll-like receptor 4), suggesting that TLR4 is a receptor of RAP and mediates its immunomodulating activity. Further analyses demonstrated that RAP rapidly activated TLR4-related MAPKs, including phosphorylated ERK, phosphorylated JNK, and phosphorylated p38, and induced translocation of NF-κB as well as degradation of IκB-α. These results are helpful to better understand the immunomodulating effects of Radix Astragali. CONCLUSIONS: RAP may induce cytokine production of RAW264.7 cells through TLR4-mediated activation of MAPKs and NF-κB.

Structure Characterization and Immunomodulating Effects of Polysaccharides Isolated from Dendrobium officinale.

A crude polysaccharide fraction (cDOP) has been determined to be the characteristic marker of Dendrobium officinale, an expensive tea material in Asia, but its chemistry and bioactivity have not been studied. In work reported here, cDOP was destarched (DOP, 90% yield) and separated into two subfraction polysaccharides, DOPa and DOPb, which were characterized by monosaccharide composition and methylation analyses and spectral analyses (FT-IR and (1)H and (13)C NMR). Both are composed of mannose and glucose at similar ratios and have a similar structure with a backbone of 1,4-linked ß-D-mannopyranosyl and ß-D-glucopyranosyl residues. Significant differences were observed only in their molecular weights. Bioassay using mouse macrophage cell line RAW264.7 indicated that DOP and its two subfractions enhance cell proliferation, TNF-α secretion, and phagocytosis in a dose-dependent manner. They also induced the proliferation of lymphocytes alone and with mitogens. DOPa and DOPb are thus proven to be major, active polysaccharide markers of D. officinale.