Study On Extraction, Sulfonation And Anticogulant Activity Of Xylan From Corn Cob

Xylan, a heterogeneous polysaccharide, which is present in the plant cell walls, also is one of the most abundant renewable resources in addition to cellulose in nature. Many literatures have reported xylan has various biological activities, such as anti-tumor, anti-oxidation and enhancing immunity. More attention is paid by people to its sulfated derivative, for more extensive biological activity and better water solubility. In this thesis, we study detailed on the extraction, sulfonation, separation and purification, physicochemical properties and anticoagulant activity of xylan, trying to select a kind of natural anticoagulant polysaccharide with potential pharmaceutical value. The main results of this study are summarized as follows:1. Xylan extraction and optimizationXylan is extracted from corn cob by using alkali formulation, Box-Behnken response surface design methodology as an approach to optimize the extraction (time, temperature, liquid-solid ratio and alkali concentration) of xylan from corn cob, in order to get the best xylan extraction process. The obtained experimental data is analysis by multiple regression to fit and statistical analysis of variance. The best extraction conditions as described below:time 90 min, temperature 70℃, liquid-solid ratio (volume ml:Quality g) 15, alkali concentration 10%(V/V). The yield was 14.75%, very close to the expected value 14.59%.2. Xylan sulfonation and optimizationBased on the contrast experiment of de-proteinization carries on the soluble xylan (WS-CCX) and insoluble xylan (WIS-CCX), we get the conclusion that the WIS-CCX effect of removing protein is better than WS-CCX, and less loss of polysaccharide, so we choose WIS-CCX to do sulfonated experiment. We optimize on the sulfonation time, temperature and the proportion of sulfonation reagent by using orthogonal design to get the best of xylan sulfonation process. By analysis of the results of orthogonal experiment, the optimum conditions as described below: sulfonated reagent ratio of 1:1 (Ⅴ/Ⅴ), time 6 h, temperature 70 ℃, the appropriate degree of substitution of 1.53. The experimental results are consistent with the orthogonal test values expected.3. Study on separation, purification and anticoagulant activity of sulfonated xylanThe separation and purification of sulfated xylan is using an anion exchange column DEAE Sepharose CL-6B, eluting with distilled water, 0.1 mol/L NaCl and 0.3 mol/L NaCl, concentrating, dialysis, and lyophilize to obtain the two main components, respectively WISX-S1 and WISX-S2, the yield was 9.09% and 63.64% respectively, where WISX-S1 is the neutral polysaccharide and WISX-S2 acidic polysaccharides. WISX-S3 was also acidic polysaccharide, but its content is only 1%. Using the activated partial thromboplastin time (APTT), prothrombin time (PT) and the thrombin time (TT) of three medical indicators to examine the anticoagulant activity of sulfonated xylan. Using physiological saline and anticoagulants heparin sodium as negative and positive controls, the results showed the two main polysaccharide components WISX-S1 and WISX-S2 can prolong APTT and memory in a certain dose-dependent manner at the experimental range. Two polysaccharide components can also extend TT, but there was no statistical significance in the PT experiment, WISX-S1 and WISX-S2 may be inferred through the endogenous and the conversion of fibrinogen into fibrin to affect coagulation pathway. In addition, anticoagulant activity of polysaccharides in vitro may have a positive correlation with sulfuric acid substitution degree and molecular weight in a certain range.4. Study on the physicochemical properties of xylanFourier transform infrared spectroscopy results showed sulfonated xylan component has typical characteristics absorption peak of sulfate groups. The gel permeation chromatograms of WISX-S1, WISX-S2 and WISX-S3 were all single peak, means that it is homogeneous polysaccharide, molecular weights of 9699 Da,12042 Da, and 5736 Da respectively. After hydrolysis of trifluoroacetic acid, polysaccharide fractions were detected by high performance liquid chromatography (HPLC), analysis results showed that WISX-S1, WISX-S2 and WISX-S3 had essentially similar monosaccharide compositions, main components were xylose, accounting for more than 90%.