| AIM: Angelica sinensis (Oliv.), also named as Danggui in China, is a natural plant of Parachute, used as an important clinical medicine in internal medicine, surgery, obstetrics, and pediatrics of Chinese medical science. Its main functions are to nourish the blood, relieve pain, enhance tonic and sthenia, activate the blood, etc.. It has been revealed that the dominantly active components of Angelica sinensis are polysaccharides, ferulic acid, volatile oil, etc., of which Angelica polysaccharides(AP) are the most abundant constituent of water soluble components and possesses wide bioactivities. Therefore, the studies on AP have become very popular in the field of researches on bioactive substances in Angelica sinensis.However, polysaccharide study is being faced with unprecedented challenges about the quality control and action mechanism since polysaccharides are a mixture and cannot penetrate the cellular membrane due to its large molecular mass. The fact that the wide bioactivities of polysaccharides have been confirmed by polysaccharide administration, such as ig, ip, iv, etc., indicates that the responsive cells might be activated via cell surface molecules(receptor). However, the molecular basis of signal transduction pathway activated by polysaccharides is not fully understood, mainly due to the lack of the identification of the specific cell-surface receptors. Toll-like receptor 4 (TLR4) is primarily activated by lipopolysaccharide(LPS) and plays an essential role in the recognition of invading pathogens, the activation of NF-kB pathway andthe production of cytokines by macrophages. It has recently been reported that TLR4 in macrophage, T cell and B cell could be activated by few bacterium polysaccharides, suggesting that TLR4 is the possible receptor of polysaccharides. Therefore, in this paper, for the first time we challenged those problems and tried to develop HPLC methods for monosaccharide composition analysis of AP and to examine whether AP can activate macrophages via TLR4.METHODS: Crude Angelica polysaccharides(CAP) were extracted and isolated from the plant materials of Angelica sinensis, purchased at Minxian County (Gansu Province, China), by the method of water boiling, ethanol precipitation, deprotein, lyophilization, etc.. Then CAP were further purified by Sephacryl S-400 chromatography into different fractions. The purity and physical-chemical properties of Angelica polysaccharides(AP) were examined by spectrophotometry, elementary analysis, specific rotation. Based on the analysis of the monosaccharide composition of AP by GC method, PMP precolumn-derivatization HPLC was further developed to identify the monosaccharide composition of AP using a C18 column and UV monitor. At the same time, peritoneal macrophages were isolated from BABL/c mice and the cellular injury model was established by treatment with hydroperoxide(H2O2) or terbutyl-hydroperoxide (tBOOH) in order to study the effects of AP on nitric oxide(NO) production of the ROS-activated macrophages and its protective actions. In addition, the immunoactivities of AP-induced cytokine production by the macrophages were investigated by the method of MTT, spectrophotometry, morphology, etc.. In this study, the effects of AP on inducible nitric oxide synthase(iNOS) mRNA and TLR4 mRNA expression in mouse peritoneal macrophages were also investigated by immunohistochemical method, RT-PCR method, etc..RESULTS: In this study, Crude Angelica polysaccharides(CAP), white powder, were well isolated by water extraction and ethanol precipitation, and the ratio of polysaccharide extraction is over 10%. In order to obtain well-distributed andstably bioactive polysaccharides, CAP were further purified by Sephacryl S-400 chromatography into three fractions, termed APF1, APF2 and APF3. The examination results of the polysaccharide purity by UV absorption, elementary analysis, specific rotation showed that there was an absence of protein and nucleic in APF1 and APF2, but there was a litter protein in APF3, suggesting that the freeze/thaw method...
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