Isolation, Purification, Structure Analysis And Bioactivities Of Armillaria Mellea Glgcoconjugates

In order to make full use of the bioactive polysaccharides from the mycelia of Armillaria mellea, AMG-IB purified from Armillaria mellea glycoconjugates (AMG) was studied. The main work include AMG extraction, purification, physical and chemical properties identification, the chang of chemical structure and hypoglycemic activity studies.1. AMG extraction, purificationThe extraction and purification procedure of AMG were as follows: Crude AMG DEAE-cellulose chromatography Sephadex G-75 column chromatography pure AMG-IB.The first step of purification was ion exchange chromatography. The solid phase was DEAE-cellulose(DE-52). The mobile phase was distilled water and NaCl 0.5mol/L. Two components AMG-1, AMG-2 were obtained.Sephadex G-75 column chromatography was used to purify AMG-1. Two components AMG-1 A and AMG-IB were separated. The purity of these two components were proved with Sepharose 4B column chromatography, FPLC and PAGE. The result showed that AMG-IB was pure component.2. chemical properties identification and structure analysisThe structure identification of AMG-1 A and AMG-IB were performed with UV spectrum, IR spectrum and gas chromatography(GC). The UV spectrum of AMG-1 A showed a character of proteid or nucleic acid in 260~280nm, but the UV spectrum ofAMG-IB didn't show that. The IR spectrum of AMG-1A and AMG-IB showed a character of polysaccharides in 4000~650cm'', and of saponin in 980cm"1,920cm"1. The component analysis of AMG-1A and AMG-IB included proteid content, polysaccharide content, uronic acid content and saponin content. The GC indicated that the polysaccharide of AMG-1A was composed of D-rhamnose, D-fucose, D-arabinose, D-mannose, D-glucose, D-galactose respectively; the polysaccharide of AMG-IB was composed of D-rhamnose, D-mannose, D-glucose respectively.3. The chang of chemical structureRelationship of AMG-IB chemical structure and bioactivity was studied. The glycan chain of AMG-IB was modified by acid hydrolysis. According to the acid hydrolysis conditions designed by test, the four condition were selected, such as 4%,40 °C ,50min; 6%,40 °C ,60min; 8%,50 °C ,40min; 10%,50°C,30min. The four fragments of acid hydrolysis had been studied on the bioactivity.The component analysis of four fragments included proteid content, polysaccharide content, uronic acid content and saponin content. The molecular weight of AMG-IB and four fragments were performed by sephrose 4B column chromatography. The Mw of the products decreased with the increase of sulphuric acid concentration.4. The study of bioactivityThe effect of AMG-1A and AMG-IB on blood glucose were studied in mice. The result showed that AMG-IB could markedly improve normal glucose tolerance. In order to reveal the relationship of structure and activity and the mechanism of hypoglycemic activity, experiments were designed from two aspects of molecular level and cell level. AMG-IB and four fragments were studied on the activity of a -glucosidase. AMG-IB, 4% and 6% fragments were able to exert glucose - lowering effect on hepatocytes. The experiment about protein nonenzymatical glycation in vitro showed that AMG-IB and four fragments had the inhibitory effect.