Separation,Structure And Anti-inflammatory Effect Study Of Polysaccharides From Smilax Glabra Roxb

PurposeSmilax glabra Roxb, also known as thorn of grifola umbellata, is a kind of plant roots can be used as medicine and as food, distribution in the Yangtze River basin and the southern provinces of China, north to Gansu, west to Yunnan province, south of Hainan, Taiwan and other places. Smilax glabra Roxb rhizoma belong to smilax genus, can remove damp-heat symptoms, detoxification, smooth joint effect. Smilax glabra Roxb is rich in starch that can be used for wine or tarts. Current the researchs are on Smilax glabra Roxb on flavonoid pharmacological study of small molecules as well as Smilax glabra Roxb process optimization, there are few studies of macromolecular substances such as polysaccharides from smilax glabra Roxb. Smilax glabra Roxb, this paper as raw material, soil extraction technology of polysaccharides from Smilax glabra Roxb, extraction, purification, characterization and study on anti-inflammatory activity and other aspects of the relationship, to develop the drug widened, also provided evidence for clinical treatment.Content and method1.This paper has been optimized optimum extraction technology of polysaccharide from Smilax glabra Roxb based on the extraction temperature, and extraction time, solid to liquid ratio and frequency of extraction of factors, based on single-factor experiment and response surface2. Amylose of Smilax glabra Roxb has been separated and purified in accordance with the best extraction process extraction of polysaccharides from Smilax glabra Roxb with ethanol alcohol, protein and pigment removal by Sevag and Sephadex gel chromatography column G-150.3. Structure characterization of polysaccharides Obtained through separation and purification polysaccharide from Smilax glabra Roxb have been preliminary studied by determination of relative molecular mass determination of monosaccharide composition4. Study on anti-inflammatory activitya. MTT assay for detection of effect of pachyman on RAW264.7macrophage growth.b. Griess assay for detection of polysaccharides from Smilax glabra Roxb on LPS-induced RAW264.7macrophages release NO impact.c. ELISA assay for detection of polysaccharides from Smilax glabra Roxb on LPS-induced effects of the release of TNF-a in RAW264.7macrophages.d. RT-PCR assay for detection of polysaccharides from Smilax glabra Roxb on LPS-induced IL-6, TNF-α in RAW264.7macrophages and effects of iNOS mRNA.e. Western Blot Assay for detection of polysaccharides from Smilax glabra Roxb on LPS-induced NF-κb in RAW264.7macrophages and effects of MAPK signaling pathways.Res u Its1. Single factors results indicates that, best extraction number for3times, best extraction temperature (℃) for90℃, best extraction time (h) for2h, best material liquid than (g/ml) for1:35, response surface analysis of extraction best technology conditions for:extraction temperature for88℃, extraction time for2.3h, material liquid than for1:36, best technology of polysaccharide have rate averages for6.41%.2.The polysaccharides of smilax glabra Roxb by the optimum extraction conditionsthat been separated to two parts tf1-1and tfl-2by ethanol alcohol, Sevag proteins, pigments and removal by Sephadex gel chromatography column G-150isolation and purification3.This paper through the determination of average molecular mass determination of monosaccharides, finds the average molecular mass of tf1-1polysaccharide average molecular mass of377Kd, tfl-2244Kd, tf1-1and tf1-2are operated by rhamnose (Rha) and galactose (Glc)2kinds of monosaccharides.4. Biological activity showed that polysaccharides from tfL-1and tfL-2had no inhibitory effect on RAW264.7macrophage growth, on LPS-induced secretion of NO and TNF-α in RAW264.7macrophages has some inhibitory effect, inhibits IL-6, TNF-α in RAW264.7macrophage cells and expression of iNOS mRNA and MAPK signaling pathway-related P-JNK, expression of P-ERK1/2protein inhibition. ConclusionThis papers through on extraction technology research of polysaccharides of Smilax glabra Roxb, got a model that can be used to forecast and analysis polysaccharides of Smilax glabra Roxb extraction technology, and derive best extraction technology of polysaccharides of Smilax glabra Roxb by the model. According to best extraction technology, through extraction, separation, and purification method, this papers got polysaccharide parts of tfL-1and tfL-2of relative molecular quality uniform, and purity high. Structure characterization research found this2species polysaccharide parts was not the same species polysaccharide. Polysaccharide parts of tfL-1and tfL-2from Smilax glabra Roxb on macrophage inhibition-free, can inhibit macrophage secretion of NO and TNF-a, has good anti-inflammatory activity, such activity may be related to inhibition of IL-6, TNF-a and iNOS mRNA gene expression and inhibition of MAPK signaling pathways associated with P-JNK, expression of P-ERK1/2related.