Active Site Tracking And Sulfated Modification Of Jujube Polysaccharide

Some researches confirmed that the biological activity or new one of natural polysaccharide can be enhanced or generated by sulfated modification. More sulfated polysaccharides can be obtained by construction reform to enhance the biological activity of Chinese herbal polysaccharides. Jujube polysaccharide (JPS) is the important biological active substances of Fructus Jujubae and has many biological activities such as enhancing immunity, anti-virus, anti-tumor, anti-aging, antioxidant, anti-inflammatory and so on. In order to track the active site of JPS and investigate the possibility of sulfated modification to improve the ativiral and immune-enhancing activity of JPS, Fractional JPSs were extracted by stepwise echohol precipitation, the best active site was selected by determination of their antiviral and imune-enhancing activity in vitro, and the further sulfated modification was carried out, the antiviral activity of polysaccharide pre- and post-modification were compared. The tests are divided into the following five sections:Test 1 Extraction of Jujube polysaccharides The total (JPSt) and four fractional JPS, JPS50, JPS60, JPS70 and JPS80 were extracted respectively by one-step and stepwise alcohol precipitation after water decoction, their polysaccharide contents were determinated by anthrone-sulfuric acid method. The results showed that the extraction rate of JPSt was the highest up to 6.59%. the extraction rate of JPS50 was 2.53% and the highest in four fraction polysaccharides.; the polysaccharide content of JPS50 was the hightest up to 52.1%.Test 2 The selection of antiviral activity site of JPS in vitro Fistly, the safety concentrations of JPS50、 JPS60、 JPS70、 JPS80 and JPSt were determined for Chicken embryo fibroblast (CEF). Then five JPSs at five concentrations within safety concentration were added into CEF monolayers by three models, pre-, post-adding polysaccharide and simultaneous adding polysaccharides and Newcastle disease virus (NDV). The effects of five JPSs on cellular infectivity of NDV were compared by MTT method. The results shows that during pre-and post-adding polysaccharides, the viral inhibition rate of JPS50 group was the highest, during simultaneous adding olysaccharides and virus, JPS70 presented highest viral inhibitory rate. The comprehensive comparison indicated that JPS50 possessed best effect and was the ativiral activity site of JPS.Test 3 The selection of immune enhancing activity site of JPS in vitro JPS50、 JPS60、JPS70、 JPS80 and JPSt at five concentrations within safety concentration were added into cultural chicken peripheral blood lymphocytes, the lymphocyte proliferation was measured by MTT method. The results showed that, JPS50 and JPS60 at 2.442-39.063 μg·mL-1 could significantly stimulate lymphocyte proliferation, JPS50 and JPS60 at 2.442-39.063μg·mL-1 could stimulate lymphocyte proliferation synergistically with PHA. The general evaluation indicated that JPS50 had the best enhance immune effect and was the immune-enhancing activity site of JPS.Test 4 The optimization of sulfated modification conditions of JPS The sulfated modification conditions were optimized taking purified JPSt as material by chlorosulfonic acid-pyridine method. L9(34) orthogonal test was designed at reaction temperature, reagent ratio and reaction time taking yield and substitution degree (DS) as index. The result showed that the effects of reagent formula and reaction temperature respectively on yield and DS were the largest. When the ratio of chlorosulfonic acid to pyridine was 1:4, the yield was the highest. When reaction temperature was 85℃, the DS was the highest. The optimum modification conditions were the reaction temperature of 85℃, ratio of chlorosulfonic acid to pyridine of 1:4 and reaction time of 1 hours in synthetical consideration.Test 5 The comparison of activiral activity of sulfated JPS in vitro The safety concentration of sJPSA, sJPS50, JPSA, JPS50 and JPSt to CEF were determined. Then five JPSs at five concentrations within safety concentration were added into CEF monolayers by three models, The effects of five JPS on cellular infectivity of NDV were compared by MTT method. The results showed that during pre-adding and simultaneous adding polysaccharides and NDV, the viral inhibitory rate of s JPS 50 and sJPSA are significantly higher than unmodified JPS. During post-adding polysaccharide, there was not significant difference among viral inhibitory rate in every group, but in sJPS50 group was highest. These indicated that sulfated modification could enhance the antiviral activity of JPS, especially sJPS50 with best effect, which was, related with DS and polysaccharide content of JPS.