| Recently medical reserch confirmed that the biological activities of many polysaccharides, especially antiviral and immunopotentiation activity, would be obtained or greatly increased after sulfated modification, which displayed a wide application prospect. Chinese angelica has been widely used in treatment of many diseses calling "nine of ten prescriptions containing it" since ancient time. As one of the major ingredient, Chinese angelica polysaccharide (CAPS) has the action of antithrombotic, immunoregulation, anti-tumor and so on. In this study, CAPS was extracted, purified and sulfated modified. The antiviral activities of four sulfated CAPSs (sCAPSs) were compared in vitro. The details are divided into three parts as follows.Test 1 Extraction, separation and purification of CAPS CAPS was extracted by water-decoction and ethanol-sedimentation method. The extractive condition, decocting temperature, ratio of drug to water and decocting time, were optimized by orthogonal design taking CAPS yield as index. The result showed the optimize condition was temperature at 100℃, ratio of drug to water being 1:20, time lasting 30min×2. After the protein was removed by Sevage’s method, CAPS was refined by chromatography with Sephadex G-200 gel to obtain two fractionated polydsaccharides CAPS1 and CAPS2. The yield and CAPS content of CAPS2 was 48.56% and 97.1%, higher than that of CAPS1 which was 9.93% and 85.2%. The protein content of CAPS2 was 0.2%, lower than 1.0% of CAPS1. Therefore, CAPS2 was as the material of sulfated modification in next experiment.Test 2 Sulfated modification of CAPS CAPS2 was modified by chlorosulfonic acid-pyr-idine method. The influences of modification condition, reaction temperature (A), reaction time (B) and the ratio of chlorosulfonic acid to pyridine (C), on the degree of substitution (DS) of product were studied by response surface methodology (RSM). Recursive model was established, the validity of the model was tested and the main effect and interaction of the factors were analysed. The results indicated that within the range of A being 65-95℃, B being 60-180 min and C being 1:3-1:14.3, the regression model of average DS (Y) relating with the three factors and was Y=2.74+0.66xA+0.66xB+0.80×C+ 0.51×A×B-0.31×A×C-0.16×B×C-0.34×A2-0.23×B2-0.50×C2.17 Sulfated CAPSs(sCAPSs) with DS ranging from 0.1230 to 4.0753 were prepared as the material of antiviral active comparison in the next experiment.Test 3 The anti-NDV activity comparison of sCAPS in vitro After the maximal safe concentrations of four sCAPSs, with DS of 1.28,1.77,2.41 and 3.22, were determined, their antiviral activities in vitro were compared by MTT method taking non-sulfated CAPS2 as control. The result indicated that sulfated modification could make CAPS obtain antiviral activities. sCAPS with DS of 3.22 had significant action in inhibiting NDV to infect. The effective concentration was within 0.1954 to 0.7813μg·ml-1。... |
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