| More and more researches had discovered that polysaccharide derivatives abtained by molecule modification can produce and enhance some biological activities. Many researches were carried out on the sulfated polysaccharide. Atractylodes macrocephala polysaccharide (AMP) is an important extraction from Atractylodes macrocephala koidz and it has many kinds of biological activities, such as immune-enhancing, anti-tumor, anti-oxidant and so on. The total Atractylodes macrocephala polysaccharide (AMPt) were extracted by one-step alcohol precipitation, and six fractional AMPs were extracted by stepwise alcohol precipitation. The best active sites were selected by determination of their antiviral and immune-enhancing activity. Then, sulfated polysaccharides were made by sulfated modification, and were compared the biological activities of pre-polysaccharide and post-modification.The tests are divided into the following five sections.Test I Extraction of Atractylodes macrocephala polysaccharide (AMP) The AMPt and six fractional AMPs, that were AMP40, AMP50, AMP6o, AMP70, AMP80, and AMP90, were extracted by water-decocting and one-step or stepwise alcohol precipitation methods, respectively. The contents of polysaccharides were determined by phenol-sulfuric acid method and protein contents were determined by coomassie brilliant blue method. The extraction rate of AMPt was the highest up to22.7%, the lowest was merely0.842%of AMP60.The content of polysaccharide of AMP50was the highest up to89.2%, the lowest was59.52%of AMP90. AMP40had the highest protein contents,10.25%, AMP80had the lowest protein contents,4.83%.Test Ⅱ The comparison of anti-virus activity of AMP The safe concentrations of every AMP on Chicken embryo fibroblast(CEF) were determined by MTT method, and AMPs at five concentrations within safe concentrations scope were added into cultivating system of CEF by three models with Newcastle disease virus(NDV), pre-, post-adding polysaccharides and simultaneous adding polysaccharide and NDV. The effects of AMP on cellular infectivity of NDV were compared by MTT method. The results showded that AMP80at3.907～7.813μg·mL-1could inhibited virus infected CEF, and its virus inhibitory rate was higher than others during pre-adding polysaccharide; All goups of AMPs at1.953～31.25μg·mL-1could inhibited NDV infected CEF significantly during post-polysaccharide, and AMP40had a higher inhibitory rate than AMP60, AMP70, AMP90, AMPt, significantly; AMP40, AMP70and AMP80at1.953μg·mL-1AMP60at3.907μg·mL-1, AMP90, AMPt at1.953～3.907μg·mL-1could inhibited NDV infected CEF significantly, and AMP70had the highest virus inhibitory rate. So, AMP40, AMP70, AMP80were selected that maybe were the optimal antiviral activity sites of AMP.Test Ⅲ The comparison of immunoenhancement of AMP Firstly, the safe concentrations of AMP40, AMP50, AMP60, AMP70, AMP80, AMP90and AMPt on chicken peripheral blood lymphocytes were determined and unified, then AMPs at five concentrations within safe concentration (including safe concentration) were adding into the chicken peripheral blood lymphocytes singly or synergistically with PHA, testing the changes of lymphocyte proliferation by MTT method. The results showed that in single adding, AMP40at31.25～62.5μg·mL-1, AMP50at500μg·mL-1, AMP70at31.25μg·mL-1250μg·mL-1, AMP80at125μg·mL-1,31.25μg·mL-1, AMP90at125～250μg·mL-1could significantly stimulate lymphocyte proliferation, and the lymphocyte proliferation rate of AMP80were higher than others. In simultaneous adding with PHA, AMP40, AMP80at31.25～250μg·mL-1, AMP60, AMP70at31.25～62.5μg·mL-1, AMP90at31.25,500μg·mL-1could stimulate lymphocyte proliferation synergistically with PHA, and AMP8have a higher proliferation rate than AMP50, AMP6o, AMP70, AMP90and AMPt significantly. The evaluation indicated that AMP80could stimulate lymphocyte proliferation singly and synergistically with PHA, and it maybe was the optimal immune-enhancing activity site of AMP.Test IV The optimization of sulfated modification conditions of AMP The sulfated modification conditions were optimized taking purified AMPt as material by Chlorosulfonic acid-pyridine method. The modification conditions were optimized by L9(3)4orthogonal design at the ratio of chlorosulfonic acid to pyridine, reaction temperature and time, taking yield, polysaccharide contents and substitution degree (DS) as index. The ratio of chlorosulfonic acid to pyridine could influence the yield significantly, and when the rate was1:6, the yield was the highest. The temperature influenced the polysaccharide contents and DS most. Polysaccharide contents was decreased as the temperature gong up, while DS was increased as temperature rising. When the reaction temperature was70℃, the yield was the highest. Taking all factors into consideration, the optimum temperature was70℃. Reaction time was not the main factor of yield, polysaccharide contents and DS, and when reaction time was1h, yield was the highest. So, the optimum modification conditions of AMP the ratio of chlorosulfonic acid to pyridine of1:6, the reaction temperature of70℃, the reaction time of1h in comprehensive consideration.Test V The comparison of anti-virus activity of sulfated AMP AMP40, AMP70and AMP80of higher antivirus activity and AMP80of higher immunocompetence were purified, sulfated modification and produced sAMP40, sAMP70, sAMP80and sAMPt (chlorosulfonic acid to pyridine of1:6, the reaction temperature of70℃, the reaction time of1h). The safe concentrations of AMPs on CEF were determined and unified. These polysaccharides at five concentrations within safe concentration were added into cultivating system of CEF by three models, pre-, post-adding polysaccharide and simultaneous adding polysaccharide and NDV. The results showed that sAMP70had higher virus inhibitory rate of71.11%than AMP40P (15.85%), AMP70P (41.09%), AMP80P (30.83%) and AMPtp (23.38%) significantly(P<0.05) during pre-adding polysaccharide. The order of virus inhibitory rates of sulfated polysaccharides was sAMP70>sAMPt>sAMP40>s AMP80. sAMP70had the highest virus inhibitory rate of62.42%than others and the virus inhibitory rate of sAMP4o(58.35%) was higher than AMP40P(35.35%) during post-adding polysaccharide. It showed that sAMP80had the highest virus inhibitory rate of88.45%than AMP40P (33.70%), AMP70P (40.40%), AMPtp (49.28%)(P<0.05) significantly and higher than AMP80P (67.92) during simultaneous adding polysaccharide and NDV. In conclusion, sulfated modification could enhance anti-virus activity of AMP, and sAMP70had the best antiviral activity.Test Ⅵ the comparison of immunoenhancement of sulfated AMP AMP40P, AMP70P, AMP80P, AMPtp and sAMP40, SAMP70, sAMP80, sAMPt at five concentrations within safe concentration scope were added into chicken peripheral blood lymphocytes, and the lymphocyte proliferation was determined by MTT method. The results showed that in single adding, lymphocyte proliferation rate of sAMP70was high up to22.4%and higher than AMP40P, AMP70P, AMP80p, AMPtp, significantly. In simultaneous adding with PHA, sulfated polysaccharide sAMP40, sAMP70, sAMP80, sAMPt and AMP80could stimulated lymphocyte proliferation synergistically, but unsulfated polysaccharide AMP40P, AMP70P, AMPt could antagonize the lymphocyte proliferation of PHA. These indicated that sulfated modification could enhance the lymphocyte proliferation rate of AMP, especially sAMP70. |
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