| Sulfated polysaccharide is a kind of the glucose hydroxyl groups with sulfate polysaccharide, and possesses many kinds of biological activities, such as antiviral activity, immune-enhancing, anti-tummor, anticoagulant, anti-aging, anti-oxidation and so on. In order to improve the biological activity of Chinese herbal medicinal polysaccharides, it may be through sulfation modification method for structural reform obtaining sulfate polysaccharide. In present research, Auricularia auricula polysaccharide was chosen as the object of study, which was extracted, purified and sulfatedly modified, and a best sulfated AAP was selected through the comparison in vitro and in vivo of antiviral and immune-enhancing activities Then the activities of it and nine kinds of sulfated polysaccharides selected by our lab were compared. The details included into ten parts as follows:Experiment 1. Preparation of sulfated Auricularia auricula polysaccharides The crude total Auricularia auricula polysaccharide (AAPct) was extracted by water decoction and one-step ethanol precipitation. Protein was then removed to obtain total Auricularia auricula polysaccharide (AAPt) by Sevage assay, which was graded into AAPi and AAP2 through column chromatography. sAAPt, sAAP1 and sAAP2 were prepared by chlorosulfonic acid-pyridine method. The polysaccharide and protein contents of sAAPs were measured by Vitriol-Anthrone and Coomassie blue G-250 meathod, respectively. Their sulphur contents were determined by Antonopoulos’ method and the structures were identified by FT-IR spectra method. The result showed that, the sulphur contents of sAAPt, sAAP1 and sAAP2 were 0.22,1.46 and 1.19, respectively. Their polysaccharide contents were 23.74%,12.60% and 10.14%, respectively. On the FT-IR spectra, there were absorptive peaks of sulfated group, which proved that the sulfated group had linked with polysaccharide to form sulfate ester.Experiment 2. Determination in vitro of antiviral activity of sAAPs Three sAAPs and threenon-sulfated AAPs dissolved with MM into 11 concentions were added into cultivating system of chick embryo fibroblast (CEF). The maximal safe concentrations of them were determined by MTT assay. Every polysaccharides at five concentrations within the safety concentration scope and Newcastle disease virus (NDV) were added into cultivating system of CEF respectively in three modes, pre-adding, post-adding polysaccharide and simultaneous adding polysaccharide and virus after mixed. The effects of cellular A570 value and virus inhibitory rate of NDV were compared. The result showed that in pre-adding polysaccharide, the A570 values of sAAP1 and sAAPt at 0.977 and 3.906 ug-mL-1, sAAP2 and AAPt at 3.906 μg·mL-1, AAP1 and AAP2 groups at 3.906～0.244μg·mL-1 were significantly higher than that of corresponding virus control group, the virus inhibitory rates of sAAPs higher than those of corresponding AAPs groups, sAAP1 was higest, the following was sAAPt group. During post-adding polysaccharide, the A570 values of sAAP1 at 0.244～3.906 μg·mL-1, sAAP2 at 1.953～0.488μg·mL-1, sAAPt at 0.488 μg·mL-1, AAPt at 1.953 μg·mL-1, AAP1 at 1.953～0.977 μg·mL-1 were significantly higher than that of corresponding virus control group, the virus inhibitory rates of sAAP1 and sAAPt group were highest and significantly higher than that of AAP2 group, which were higher than that of AAPs. During simultaneous adding polysaccharides with virus after mixed, sAAPs group at 1-5 concentration and AAPs with two concentration were significantly higher than that of corresponding virus control group, the viral inhibitory rates of sAAP 1 was highest. The viral inhibitory rates of sAAPs groups were significantly higher than that of AAPs group. Theses results indicated that sAAPs at the suitable example-adding modes and doses could significantly inhibit NDV to infect CEF, sulfated modification could enhance the antiviral activity of AAP. In general evaluation the actions of sAAP1 and sAAPtwere stronger.Experiment 3. Determination in vitro of immune-enhancing activity of sAAPs. Three sulfated sAAPt, sAAP1, sAAP2 and three non-sulfated AAPt, AAPi, AAP2 were dissolved with MM into five concentions within safety concentration and added into the cultured chicken peripheral blood lymphocyte in single or simultaneous with PHA, respectively, the changes of the lymphocyte A570 value and the lymphocyte proliferation rate were determined by MTT method. The results showed that in single adding, the A570 values of sAAPt and sAAPigroup at 1.953～0.244 μg·mL-1, sAAP2 at 0.024μg·mL-1, AAPt and AAP2 group at 0.488 μg·mL-1 were significantly higher than that of corresponding cell control group, the lymphocyte proliferation rates of sAAPs higher than those of AAPs groups, sAAP1 was highest and following was sAAPt group. In simultaneous adding with PHA, the A570 values of sAAP1 at 1.953～0.244 μg·mL-1, sAAPt and sAAP2 at 0.244μg·mL-1, AAP1 group at 1.953～0.244μg·mL-1 were significantly larger than that of corresponding PHA control group, the lymphocyte proliferation rates of sAAPs were higher than that of AAPs group, sAAP1 was highest, following was sAAPt group. These results indicated that sulfated modification could enhance the immune-enhancing activity of AAP. In general evaluation the actions of sAAP1, sAAPt were stronger.Experiment 4. Determination in vivo of antiviral activity of sAAPs determination Three hundred 26 days old chickens were divided randomly into 6 groups. the chickens except blank control group were attacked with intramuscular injection of New castle disease virus (NDV), After attacked for 6 hours, the chickens in two sAAPs and two AAPs group were intramuscularly injected respectively with 2mg per chick of sAAPt, sAAP1,AAPt and AAP1, in control group, physiological saline, once a day for three successive day. Then the clinical signs and death were mainly recorded everyday for 14 successive day. Mortality rate, cure rate and total effective rate was statisticed. Before attack one day and on days 3,7,14 after attack, the blood samples were collected from brachial vein for determination of serum HI antibody. The results showed that the mortalities of all polysaccharide group were lower and the total effective rates were higher than those of challenge control group. In sAAPt group, the mortality was lowest mortality, and cure rate and total effective rate were highest. After challenge the antibody titers of all polysaccharide groups were higher than those of virus control group, the antibody titers of sAAPt at all time points were highest, and significantly higher than those of challenge control group. Those indicated that all polysaccharides possessed determinate curative effect on artificially infected ND, sulfated modification can further enhance the AAP in vivo antiviral activity, and the efficacy of sAAPt was the best.Experiment 5. Determination in vivo immune-enhancing activity of sAAPs determination One hundred and eighty 14 days old chickens were randomly divided into six groups, sAAPt, sAAP1, AAPt, AAP1, no adjuvant (only vaccination, NA) and blank control group (BC). The average titer of maternal antibody in 14 days old chicken was 3.2 log2. All group except blank control were vaccinated with Newcastle disease vaccine and the exercise repeated at 28days old. At the same time of the first vaccination, the chickens in groups 1-4 were intramuscularly injected respectively with 0.5 mL of corresponding polysaccharides, in NA and BC group with 0.5 mL of physiological saline, once a day for three successive days. On days 7 (D7),14 (D14),21 (D21) and 28 (D28) after the first vaccination, four chickens were sampled randomly from each group for determination of peripheral lymphocytes proliferation by MTT assay and six chickens were sampled randomly from each group for examination of serum hemagglutination inhibition (HI) antibody titer by micro-method. The results showed that, all time points after the first vaccination, the serum antibody titers and promote lymphocytes proliferation of sAAPt and sAAP1 were significantly higher than that of NA group, and the promote lymphocyte proliferation of sAAPs were significantly higher than those of AAPS. The promote lymphocyte proliferation of sAAPt was highest, following was sAAP1 group. Those results indicate that sAAPs could could significantly enhance antibody titer and promote lymphocyte proliferation of AAP. In general evaluation the efficacy of sAAPt was best, following was sAAP1.Experiment 6. Comparison in vitro of antiviral activity of ten kinds of sulfated polysaccharides Selected sAAPt based on above-mentioned determination result and sulfated Astragalus Polysaccharide (sAPS), sulfatied Epimedium polysaccharides (sEPS), sulfated Lentinan polysaccharide (sLNT), sulfated Chinese Angelica polysaccharides (sCAPS2.2), sulfated Lycium barbarum polysaccharides (sLBPS1.5), sulfated Tremella polysaccharide (sTPS7oc), sulfated Codonopsis pilosula polysaccharides (sCPPS50c), sulfated Siberian solomonseal rhizome polysaccharide (sSRPSso), sulfated Achyranthes root polysaccharide (sARPSt) were selected based on the previous determination result of antiviral activity test, which were comparised. Firstly, the safe concentrations of ten sulfated polysaccharides to CEF were determined by MTT method. Ten polysaccharides at five concentrations within safe concentration were then added into the cultivating system of CEF in three models (pre-, post-adding polysaccharides and simultaneous adding polysaccharides and NDV after mixed), the cellular infectivity of NDV were observed by MTT assay. The result showed that in pre-adding polysaccharide, the A570 values of sAPS and sTPS70c at 0.244 μg·mL-1, sEPS at 0.488 μg·mL-1,sSRPS50 at 0.977 and 0.244μg·mL-1 were significantly higher than that of corresponding virus control group, the virus inhibitory rates of sAAPt and sSRPS50 was highest and significantly higher than those of sLNT and sLBPS1.5, following was sEPS, sAPS, STPS70c, sCAPS2.2, sCPPS50c and sARPSt group. During post-adding polysaccharide, the A510 values of sEPS at 0.977～0.488 μg·mL-1, sAAPt at 1.953～0.244μg·mL-1 sSRPS50 at 0.488～0.244 μg·mL-1, sCAPS2.2 at 0.977 μg·mL-1; sAPS, sTPS70c and sLNT at 0.244 μg·mL-1 were significantly higher than that of NDV group, the virus inhibitory rates of sEPS was highest, which was significantly higher than that of sARPSt, sLBPS1.5 and sLNT group, following were sAAPt, which was significantly higher than that of sLNT, then was sAPS, sSRPS50, sCAPS2.2and sCPPSSOc group. During simultaneous adding polysaccharides with virus after mixed, the A570 values of sAPS, sSRPS50 and sAAPt at 0.244μg·mL-1 sTPS70c at 0.488～0.244 μg·mL-1 were significantly higher than that of corresponding virus control group, the virus inhibitory rates of sEPS was highest, which was higher than that of sLNT and sLBPS1.5, following were sAPS,sAAPt, sSRPS50, sCPPS50c and sCAPS2.2. Theses results comparison indicated that sEPS was the best anti NDV activity in the invitro test, following was sAAPt, sAPS, sCPPS50c, sSRPS50 and sCAPS2.2 group.Experiment 7. Comparison in vitro of immune-enhancing activity of ten kinds of sulfated polysaccharides Ten kinds of above-mentioned sulfated polysaccharies were dissolved with MM into five concentions within safety concentration and added into the cultured chicken peripheral blood lymphocyte in single or simultaneous with PHA, respectively. Used MTT method to deremined the changes of the lymphocyte A570 value and the lymphocyte proliferation rate. The results showed that in single adding, the A570 values of sEPS, SCPPS50c,sSRPS50 and sAAPt at 1-5 concentration groups, SCAPS2.2 and sAPS had four concentration, sLBPS1.5 and sTPS70c at 0.244 μg·mL-1 were significantly higher than that of corresponding cell control group, the lymphocyte proliferation rates of sEPS, sAAPt were highest, which were significantly higher than that of other group. Following were sAPS, sCPPS50 and sSRPS50 group, which were significantly higher than those of sLBPS1.5, sTPS70c sLNT and sARPSt group. In simultaneous adding with PHA, the A570 values of sLNT at 0.488 μg·mL-1, sAAPt at 0.244 μg·mL-1 significantly higher than that of corresponding PHA control group, the lymphocyte proliferation rates of sAAPt was highest, which was higher than those of other nine group. Following were sEPS, sAPS, sCAPS2.2, sSRPS50 and sCPPS50c group. The comprehensive comparison results indicated that sulfated modification could enhance the immune-enhancing activity. In general evaluation the actions of sEPS, sAAPt, sAPS, sCPPS5oc, sSRPS50 and sCAPS2.2 were stronger.Experiment 8. Comparison in vivo of antiviral activity of six kinds of sulfated polysaccharides The stronger antiviral activity of 6 kinds of sulfated polysaccharides (sEPS, sAPS, sAAPt, sCAPS2.2,sCPPS5oc and sSRPS50) were chosen by the comparison in vitro test. Four hundred and eight 22 days old chickens were divided randomly into 8 groups, the chickens except blank control group were attacked with intramuscular injection of New castle disease virus (NDV), After attacked for 6 hours, the chickens in 6 sulfated polysaccharides were intramuscularly injected respectively with 2mg for each drug, in control group, physiological saline, once a day for three successive day. Then the clinical signs and death were mainly recorded everyday for 14 successive day. Mortality rate, cure rate and total effective rate was statisticed. Before attack one day(D-1) and on days 3 (D3),7 (D7) and 14 (D14) after attack, the blood samples were collected from brachial vein for determination of serum HI antibody. The results showed that the mortalities of all polysaccharides group were lower and the total effective rates were higher than those of challenge control group.The mortality of sEPS and sAAPt group was lowest, and cure rate and total effective rate were highest. After challenge the antibody titers of all polysaccharide groups were higher than those of virus control group, the antibody titers of sEPS and sAPS group on D3, D7,Di4 significantly higher than that of challenge control group. These results indicate that all polysaccharides possessed determinate curative effect on artificially infected ND, and the efficacy of sEPS and sAAPt was the best.Experiment 9. Comparison in vivo of immune-enhancing activity of six kinds of sulfated polysaccarides The immune-enhancing activities in vivo of sEPS, sAAPt, sAPS, sCPPS5oc, sSRPS50 and sCAPS2.2 which were screened by the test in vitro and had better immune-enhancing effects, were further compared. Two hundred and fourty 14 days old chickens were divided randomly into 8 groups. The chickens except blank control group were vaccinated with ND vaccine, repeated vaccination at 28 days old. At the same time of the first vaccination, the chickens in six sulfated polysaccharides groups were respectively injected with 2.0 mg for each drug, in No adjuvant (NA, only vaccination) and blank control (BC) group, with physiological saline, respectively, once a day for three successive days. On days 7,14,21 and 28 after the first vaccination, the changes of serum HI antibody titer and peripheral lymphocyte proliferation were determined. The result showed that the antibody titer of sEPS and sAAPt group at every time point were highest, which were significantly higher than that of NA group, sCAPS2.2 on days 14,21 and 28, sAPS and sSRPS50 group on days 14 and 28 were significantly higher than that of corresponding NA group. The The A570 value of sEPS on days 14,21 and 28, sCPPS50c on days 21, sAAPt group on day 28 after the first vaccination were significantly larger than that of corresponding NA group. The lymphocyte proliferation rates of sEPS was highest, following was sAAPt group. These results indicated that six sulfated polysaccharides could significantly promote the immune response of ND vaccine. In general evaluation the actions of sEPS and sAAPtwere stronger.Experiment 10. Effects of two kinds of sPSs on expression of IFN-y mRNA of chicken peripheral lymphocyte In order to further study the molecule mechanism of sAAPt and sEPS in immune-enhancing actions, sAAPt, sEPS and AAPtat three concentrations (3.91,1.95, 0.98μg·mL-1) were added into the chicken peripheral blood lymphocyte and cultured 12 h, the cells were harvested and total RNA was isolated, the mRNA expression of IFN-y was evaluated by fluorescence quantitative RT-PCR assay. The results showed that sAAPt at 0.98 μg·mL-1, sEPSH at3.91μg·mL-1 could promote the expression of IFN-y mRNA, which significantly stronger than PHA and AAPt and there was a certain of dose-efficacy relationship. |
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