| Newcastle disease virus (NDV) is seriously threatened to the poultry industry developing through out world. At present, the most effective control method is vaccination for prevention and no curative drugs were developed.According to the results of the screening various polysaccharid in chinese traditional herbs, which have the anti-activity to NDV, the following studies with Epimedium polysaccharide were carried ous:The L933orthogonal design was hired to investigate extraction conditions of hypersound extraction and hypersound combined with enzyme extraction based on single-factor studied. The optimizal condition for hyperacoustic extraction was as following: duration time was 50 min while the ratio of dry alage to water was 1∶20 in 60℃. in this condition, the extract rate of polycose extracted from epimedium was 24.12% measured by phenol- sulphuric acid method, and the content was 0.56%; the optimizal extract condition of hypersound combined with enzyme extraction was as follws: the content of enzyme was 0.210g, while the duration was 30 min in pH 9 and 70℃; the extract rate of EPS was 45.23%, the content was 1.06%. The results indicated that the method of hypersound combined with enzyme extraction was superior to hypersound extraction. Repeated freezing and thawing combining Sevag was the best method among Sevag, repeated freezing and thawing, repeated freezing and thawing combining Sevag in deproteinization. Two eluting peakes were observed when EPS was separated by DEAE-52.Conditions for sulfated modification of EPS were optimized by L933orthogonal design through the index of SO42- content and DS based on volume ratio of chlorosulfonic acid and pyridine, reaction time and temperature. The best condition for sulfating was as follows: the volume ratio was 1∶6, while reaction time was 2.5h in 60℃; in this condition, the content of SO42- in polycose was 13.57%, and DS was 1.22. The results of ultraviolet spectra indicated that there was a strong absorption peak between 190～220nm, which was the absorption peak of polyose, and there was a visible characteristic absorption peak of SO3 between 250~275nm. The analysis of infrared spectra (IR) indicated that sulfated esterification was successful, EPS and sEPS both had the feature absorption peak of polysaccharide, besides sEPS also had the feature absorption peak of S=O under close to 1259cm-1 and the feature absorption peak of C-O-Sunder in about 787cm-1.MTT results of cell culture in vitro showed that the anti-NDV activity of sEPS was better than that in EPS, especially the method of pre-adding polysaccharide and simultaneous adding mixed polysaccharides with NDV, at first, EPS'anti-NDV was weak, however, the acti-NDV activity was greatly enhanced after sulfated, sEPS played a crucial role in preventing adsorbtion of NDV to host cell, and it also disturbed the duplication of virus, therefore, it could be against the virus directly.We aimed to find a proper method for deproteinization by optimizing the extraction of EPS, and optimize the sulfated modification and their conditions, the mechanism of EPS in anti-NDV was further probed. Study on extraction method and conditions of EPS, found the best method of getting rid of protein, optimized the sulfated modification conditions, compared the antiviral of EPS to sEPS, and approach the antiviral mechanism in NDV. That had provided theoretical foundation for polysaccharide and sulfated polysaccharides (SPS) in Chinese traditional herbs in clinical preservation and therapeutics... |
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