| Polysaccharide is a kind of macromolecular compound existing in nature widely and is an important bioactive substance in life-body. It could be involved in lots of life phenomenon. Because of the extensive pharmacological function, for example of enhancing immune, antiviral, antioxidation, anti-aging and so on, polysaccharides attracted many researchers’ attention. Now, researches had discovered that some natural polysaccharides could get more extensive application after sulfated. Isatis root polysaccharide (IRPSt) is one of an important extraction from Radix Isatidis, and it has many kinds of biological activities, such as immune-enhancing, antiviral and so on. The total Isatis root polysaccharide (IRPSt) was extracted by water-decocting and one-step alcohol precipitation method. Three fractional polysaccharides, IRPSi, IRPS2 and IRPS3, are got through separation of Sephadex G-100. The best active sites were selected by determination of their antiviral and immune-enhancing activity. Sulfated polysaccharides were made by sulfated modification. Then, a sulfated polysaccharide would be selected for its better biological activities. The tests are divided into the following six sections.Test I Extraction and purification of Isatis root polysaccharide (IRPS) The total Isatis root polysaccharide (IRPSt) was extracted by water-decocting and one-step alcohol precipitation method. Three fractional polysaccharides, IRPSi, IRPS2 and IRPS3, are got through separation of Sephadex G-100. Their polysaccharide content were determined by phenol-sulfuric acid method. The results showed that the exctration rate of IRPSt was up to 10.01%, and its content was 84.92%. The content of polysaccharide of IRPS1 was the highest, reached 96.61%, the lowest was 40.94% of IRPSs.Test II The screening of antivirus active site of IRPS The safe concentrations of IRPSt, IRPS1, IRPS2 and IRPS3 on Chicken embryo fibroblast (CEF) were determined firstly, and five concentrations within safe concentrations scope of theirs were added into cultivating system of CEF by three models with Newcastle disease virus (NDV), pre-, post-adding polysaccharides and simultaneous adding polysaccharides and NDV. The A570 of cells were determined by MTT assay to evaluate the effects of these polysaccharides on the virus infecting the cell. The results showed that four polysaccharides (IRPSt, IRPS1, IRPS2 and IRPS3) could inhibited NDV infected CEF in some degree in the three models, and IRPSt had the best virus inhibitory rate, and the virus inhibitory rate of IRPS2 was higher than those of IRPS] and IRPS3 in the three fractional polysaccharides.Test Ⅲ The screening of immune-enhancing active site of IRPS The four polysaccharides (IRPSt, IRPS1, IRPS2 and IRPS3) 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 assay after 44 hours. The results showed that in single adding, all of four polysaccharides could stimulate lymphocyte proliferation at suitable dosages. In simultaneous adding with PHA, IRPS1 and IRPS2 could stimulate lymphocyte proliferation-. And the lymphocyte proliferation rate of IRPS2 was the highest no matter singly or synergistically with PHATest IV The sulfation modification of IRPS IRPS2 were modified by sulfation modification of chlorosulfonic acid-pyridine method. The modification conditions were optimized by L9(3) orthogonal design taking the ratio of chlorosulfonic acid to pyridine, reaction temperature and time as factors. The nine sulfated polysaccharides were prepared named sIRPS1, S1RPS2, SIRPS3, SIRPS4, sIRPS5, siRPS6, sIRPS7, sIRPS8 and sIRPS9. The results showed that the reaction temperature of 75-85℃, ratio of chlorosulfonic acid to pyridine of 1:8-1:6 and reaction time of 2-3 hours profit the substitution of sulfation group.Test V The comparison of antivirus activity of sulfated IRPS The safe concentrations of nine sulfated polysaccharides (sIRPS1-SIRPS9) and IRPSt 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 cellular infectivity of NDV was determined by MTT assay. The results showed that during pre-adding polysaccharide, the antiviral activities of sIRPS1, SIRPS2, SIRPS3, sIRPS4, SIRPS5, sIRPS7, sIRPS8, sIRPS9 and IRPS, were significantly higher than that of sIRPSs; during post-adding polysaccharide, the antiviral activities of IRPSt, IRPS2, IRPS4, IRPS7, IRPSg and IRPS9 were significantly higher than those of IRPSh IRPS3, IRPS5 and IRPS6; during simultaneous adding polysaccharide and NDV, the antiviral activities of IRPS4, IRPS7 and IRPSs were significantly higher than those of other groups. The antiviral activity of IRPS4 was the highest. It suggested that sulfation modification could enhance antivirus activity of IRPS and SIRPS4 had the strongest activity.Test VI the comparison of immune-enhancing activity of sulfated IRPS The nine sulfated polysaccharides (sIRPS1-sIRPS9) and IRPSt at five concentrations within safe concentration were added into the chicken peripheral blood lymphocytes singly or synergistically with PHA, the changes of lymphocyte proliferation significantly by MTT assay. The results showed that during single stimulation,10 polysaccharides could stimulate lymphocyte proliferation; during synergistical stimulation with PHA, SIRPS4, sIRPSs, sIRPS6 and sIRPS7 could stimulate lymphocyte proliferation. The lymphocyte proliferation rate of SIRPS4 was the highest no matter singly or synergistically with PHA. It suggested that sulfation modification could enhance antivirus activity of IRPS, SIRPS4 had the strongest activity and its modification condition could be as optimal modification condition of IRPS that is the reaction temperature of 75℃, ratio of chlorosulfonic acid to pyridine of 1:4 and reaction time of 2 hours. |
PDF Full Text Request