Studies On The Extraction, Purification,Structure And Part Of Bioactivities Of Polysaccharides From Gleditsia Sinensis Lam

The mature pod of Gleditsia sinensis Lam. which is Legum inosae, the main chemical compositions of saponins, sterides, flavonoids, polysaccharides, and so on, But only saponins were utilized while so much polysaccharides were seldom researched. In recent years, many researches indicated that some polysaccharides has antihepatotoxic, regulation of the immune system, antioxidant activities and has effects on the central nervous system. For the rational development and utilization of this valuable resource, the extraction, isolation, purification, composition analysis, molecular weight, structure, anti-diabetic activity and antibacterial were systematically studied. The main contents and results are as follows:1. The four factors including temperature, time, solid-liquid ratio, and the extraction times on the yield of the polysaccharides from the mature pod of Gleditsia sinensis Lam. were studied by orthogonal experimental design, on the basis of single factors tests. The results showed that solid-liquid ratio was the most important factor for the extraction percentage of the polysaccharides, and the extraction time was second. The optimal extraction technology conditions were that the solid-liquid ratio was1:45, the temperature50℃, the extraction time70min and the extraction times twice. The yield of the polysaccharides of Gleditsia sinensis Lam.(GSP) was35.46%. GSP was removed soluble substances and partially denatured protein first by85%eheanol alcohol; then different concentrations of eheanol were used to grade the polysaccharides, so that the polysaccharides in ehanol concentrations30%,45%,70%,85%were centrifuged respectively, the polysaccharides were divided into five grades. The70%ethanol alcohol precipitation of GSP was removed protein with Sevag method, GSP-1, GSP-2, GSP-3, GSP-4were achieved by further purified with hollow fiber ultrafiltration membrane. Then GSP-1, GSP-2and GSP-3were examined as homogeneous polysaccharides by HPGPC and Sephadex G-200gel column, and the molecular weight were4.69×105,3.96×105,3.18×105.2. The infrared spectras of GSP showed typical characteristic absorption peaks, and the structure of GSP-1, GSP-2, GSP-4was pyranoid glucan with β-glycosidic bond. GSP-1, GSP-2, GSP-3and GSP-4were hydrolyzed by2M sulphuric acid, then their components and structures were measured by HPLC. The results showed that GSP-1was mainly composed of rhamnose, xylose, arabinose and galactose, the molar ratio was5.34:2.65:45.13:46.88; GSP-2was mainly composed of rhamnose, xylose, arabinose, mannitose and galactose, the molar ratio was4.96:14.51:18.27:5.04:57.22; GSP-3was mainly composed of rhamnose, xylose, arabinose,mannitose and galactose, the molar ratio was5.50:6.17:5.84:5.98:76.50; GSP-3was mainly composed of xylose, arabinose, mannitose and galactose, the molar ratio was3.56:1.82:13.75:80.87. Periodic acid oxidation results showed, GSP-1, GSP-2, GSP-3, GSP-4containing1�'6glycosidic bonds,1�'2and (or)1�'4glycosidic bonds, where in1�'2and (or)1�'4glycosidic bonds may be polysaccharides backbone sugar residues in the link. The experiment of Congo red showed that GSP-1, GSP-2, GSP-3, GSP-4were with triple helix stereo-configuration possibility.3. Based on the reduction ability, it was concluded that, taking Vc as reference, the reduction ability of GSP-1, GSP-2, GSP-3, GSP-4were weaker than Vc, and the reduction ability of samples from strong to weak as:GSP-2>GSP>GSP-3>GSP-4: The abilities of the Polysaccharides to scavenge hydroxyl radicals was tested by phenanhroline-Fe2+-H2O2reaction, and hydroxyl scavenging from strong to weak were as followed:GSP>GSP-3>GSP-1>GSP-4>GSP-2, the scavenging CC50on·OH were0.3790mg/ml,0.4449mg/ml,0.6550mg/ml,0.7660mg/ml and1.0407mg/ml. The abilities of the polysaccharides to eliminate O2-was determined through Pyrogallol auto oxidation, the inhibition effect on O2-·of the polysaccharides were obvious, especially for GSP-2、GSP and GSP-1, GSP-3and GSP-4are less significant, and the inhibition IC90were2.2242mg/ml,2.5622mg/ml,2.0618mg/ml,2.0435mg/ml and3.0378mg/ml. The scavenging effect on NO2-of the polysaccharides were significant, and with a great relation on concentration, The scavenging effect from strong to weak generally the order:GSP and GSP-3> GSP-2>GSP-1and GSP-4, the scavenging CC50to NO2-were0.1527mg/ml,0.1482mg/ml,0.2920mg/ml,0.3483mg/ml and0.4057mg/ml. The result of antibacterial activity of the polysaccharides were ineffective, GSP on Gram-positive bacteria inhibition were stronger(eg. the minimum inhibitory concentration of Staphylococcus aureus:GSP is75mg/ml, GSP-1is75mg/ml, GSP-2is50mg/ml and GSP-4is75mg/ml), and (?)gainst Gram-negative bacteria had a certain effect(eg. the minimum inhibitory concentration of Escherichia coli:GSP was75mg/ml, GSP-1was50mg/ml, GSP-2was75mg/ml and GSP-4had no inbitition) in100mg/ml following concentration range, GSP had no inbitition on fungus in100mg/ml following concentration range.