| Polysaccharide is a class of important biomacromolecules in organism like proteins and nucleic acids, and possesses many kinds of biological activities, such as immune-enhancing, anti-tummor, anti-aging, anti-oxidation and so on. Many studies confirmed that sulfated modification could further enhance the biological activity of polysaccharide and produce new pharmaceutical value, especially in enduing or reinforcing better antiviral and immunostimulatory effects. In this research the total Tremella polysaccharide (TPS) and three fractional TPSs were extracted by one-step or stepwise ethanol precipitation method respectively. Their antiviral and immune-enhancing activities were compared and two TPSs with better efficacy were selected. The sulfated modification conditions of TPS were optimized by orthogonal experiment, with which two TPSs were modified to obtain two sulfated TPSs (sTPSs). Meanwhile, Two Codonopsis pilosula polysaccharides (CPPSs) selected by our lab were modified with the optimized conditions selected by our lab to obtain two sulfated CPPSs (sCPPSs). The antiviral and immune-enhancing activities of these four sulfated polysaccharides (sPSs) were compared. The details are divided into nine parts as follows:Experiment 1 Extraction of Tremella polysaccharide The crude total TPS (TPStc) and three crude fractional TPSs, TPS60c TPS70c and TPS80c, were extracted by water-decocting and one-step or stepwise ethanol precipitation method, respectively, after tremella was wetly ground and the cell wall was broken with supersonic waves. The carbohydrate and protein contents of all polysaccharides were determinated recpectively by phenol-sulfuric acid and coomassie brilliant blue G-250 methods. The results showed that extraction rate of TPStc was hightest up to 6.39%. Among three fraction polysaccharides, extraction rate of TPS70c was highest,3.86%. The carbohydrate content of TPS70c was highest,86.23%. The protein contents of TPStc and three fractional TPSs were lower. The results indicated that wet grinding and ultrasonic treatment for Tremella fuciformis could enhance the extraction rate of TPS.Experiment 2 The effect of TPS on cellular infectivity of NDV in vitro In order to select the antiviral active site of TPS, Firstly The safe concentration of TPStc, TPS6Oc TPS70c and TPS80c on Chicken embryo fibroblast (CEF) were compared by MTT method. Then four TPSs at five concentrations within safe concentration and NDV were added into cultivating system of CEF in three models, pre-, post-adding polysaccharide and simultaneous adding polysaccharides and NDV after mixed.The effects TPS on cellular infectivity of NDV (the A570 value and highest viral inhibitory rate) was comared by MTT method.The results showed that four TPSs at 3.907～62.5 μg·mL-1 in three sample-adding modes significantly inhibited NDV infection of CEF. During pre-adding polysaccharide the virus inhibitory rate of TPS80c at 62.5 μg·mL-1 group was highest, during post-adding polysaccharide, TPStc at 3.907 μg·mL-1 group, and during simultaneous adding polysaccharides and NDV after mixed, TPS60c at 3.907 μg·mL-1 group. In comprehensive comparison TPStc presented best action and maybe was the antiviral active site of TPS.Experiment 3 The effect of TPS on chicken peripheral lymphocyte proliferation in vitro In order to select the immune-enhancing active site of TPS, four TPSs at five concentrations within safe concentration were added into the cultivating system of peripheral blood lymphocyte singly or synergistically with PHA. The peripheral blood lymphocyte proliferation was determined by MTT method (A570 value and hightest lymphocyte proliferation rate). The results showed that in single adding TPS70c at 3.907～62.5 μg·mL-1 and TPStc at 31.25 μg·mL-1 could significantly stimulated lymphocyte proliferation, and the lymphocyte proliferation rate of TPStc at 31.25 μg·mL-1 was highest. In simultaneous adding with PHA TPS70c at 15.625～31.25 μg·mL-1, TPStc at 15.625 μg·mL-1 could significantly stimulated lymphocyte proliferation. The lymphocyte proliferation rate of TPS70c at 15.625 μg·mL-1 was highest. In general evaluation, TPS70c possessed best action and maybe was the immune-enhancing active site of TPS.Experiment 4 Purification and sulfated modification condition optimization of TPS TPStc was purified by chromatography through DEAE-Sepharose Fast Flow and Sephadex G-200 column in turn to get purified TPS (TPStp). The modification conditions of chlorosulfonic acid-pyridine method, reagent formula, reaction temperature and reaction time, was optimized by L9(34) orthogonal test taking the degree of substitution (DS) and yield as index. The result shows that the carbohydrate content of TPS was increased after purified. The effects of reaction temperature on DS and yield were greatest, when reaction temperature was 80℃, DS and yield were highest (1.62 and 512.4 mg respectively). In synthetical consideration the optimum modification conditions were the ratio of chlorosulfonic acid to pyridine of 1:6, reaction temperature of 80℃ and reaction time of 1.5 hours.Experiment 5 Effects of two kinds of sPSs on cellular infectivity of NDV in vitro The sulfated modification of TPStp and TPS70c were performed under the optimized conditions to obtain two sulfated TPSs, sTPStp and sTPS70c.Meanwhile, two CPPSs, CPPStp and CPPS50c, were modified with the optimized conditions selected by our lab to obtain two sulfated CPPSs, sCPPStp and sCPPS50c. Firstly the safe concentration of four sPSs and non-modified TPStp on CEF were determined by MTT method. Then five polysaccharides at five concentrations within safe concentration with NDV were added into the cultivating system of CEF in three models, pre-, post-adding polysaccharide and simultaneous adding polysaccharides and NDV after mixed.The effects of five polysaccharides on cellular infectivity of NDV were comared by MTT method (A570 value and the hightest virus inhibitory rate). The results showed that five polysaccharides at 0.391～6.25 μg·mL-1 in three sample-adding modes significantly inhibited NDV to infect CEF. In pre-adding polysaccharide, the virus inhibitory rate of sTPStp at 1.563 μg·mL-1 group was highest, in post-adding polysaccharide, sTPStp at 1.563 μg·mL-1 group, and in simultaneous adding polysaccharides and NDV after mixed, sCPPS50c at 6.25 μg·mL-1. These results indicated that sulfated modification could enhance the antiviral activity of polysaccharides, sTPStp presented strongest action and the next was STPS70c, which was related to the DS at a centain extent.Experiment 6 Effects of two kinds of sPSs on chicken peripheral and splenic lymphocyte proliferation In order to compare the immune-enhancing action in vitro four sPSs, four sPSs and non-modified TPStp at five concentrations were added into the cultivating system of chicken peripheral or splenic lymphocyte singly or synergistically with PHA or LPS. The lymphocyte proliferation (A570 value and hightest lymphocyte proliferation rate) was determined by MTT method. The results showed that in single adding into peripheral lymphocyte, four sPSs almost at 5 concentration groups and TPStp only at 3.125 μg·mL-1 could significantly stimulate lymphocyte proliferation and the proliferation rate of sCPPStp at 0.391 μg·mL-1 was the highest. In simultaneous adding into peripheral lymphocyte with PHA, sCPPS50c at 0.391～1.563 μg·mL-1, sTPStp at 0.782 μg·mL-1 and sCPPStp at 125 μg·mL-1 significantly stimulated lymphocyte proliferation and the proliferation rate of sCPPS50c at 0.782 μg·mL-1 was the highest. In single adding into splenic lymphocyte, four sPSs at 3 to 5 concentration groups and TPStp only at 3.125 μg·mL-1 significantly stimulate splenic lymphocyte proliferation, and the proliferation rate of sCPPStp at 3.125 μg·mL-1 was highest. In simultaneous adding into splenic lymphocyte with LPS, five polysaccharides at 6.25 μg·mL-1, STPS70c at 0.781 μg·mL-1 and sCPPStp at 3.125 μg·mL-1 significantly stimulated lymphocyte proliferation, and the proliferation rate of STPS70c at 0.781 μg·mL-1 was the highest. These results indicated that sulfated modification could enhance the immune-enhancing action of polysaccharide, the action of sCPPS50c was strongest, which was related to the DS at a centain extent.Experiment 7 Effects of two kinds of sPSs on immune response of ND vaccine in chickens In order to compare the immune-enhancing action of four sPSs in vivo, the effects of the polysaccharides on immune response of ND vaccine in chickens were determined.476 14-day-old chickens were randomly divided into 17 groups and vaccinated with ND vaccine except for blank control (BC) group, repeated vaccination at 28-day-old. At the same time of the first vaccination, the chickens in 6 sTPS groups and 6 sCPPS groups were intramuscularly injected respectively with sTPStp、STPS70c、sCPPStp and sCPPS50c、at high, midum and low dose, in 3 TPS control groups, TPStp, in non-adjuvant control (NA) group, physiological saline. On days 7,14,21 and 28 after vaccination, the blood samples were collected from brachial vein for determination of serum HI antibody, form cardiopuncture for determination of peripheral T lymphocyte proliferation. The results showed that four sPSs at suitable dose and some time points significantly raise antibody titer and promote T lymphocyte proliferation, while TPStp did not present obvious action. In general evaluation, the action of STPS70c at midum dose was best, the next is sCPPS50c at midum dose.Experiment 8 Comparison of curative effects of two kinds of sPSs for ND in chickens In order to compare the antiviral activity of two kinds of sPSs in vivo, the curative effects of four sPSs for ND of artificial infection in chicken was determined.245 27-day-old chickens were divided randomly into 7 groups and challenged with NDV except for blank control (BC) group. After 6 hours of challenge, the chickens in four sPSs and polysaccharide control group were intramuscularly injected respectively with sTPStp, STPS70c, sCPPStp, sCPPSsoc and TPStp, in challenge control and BC group, physiological saline, once a day for three successive day. Then the clinical symptoms and death status were observed daily. On day 14 after challenge, the mortality, cure rate and total effective rate was calculated. On day 1 before challenge and days 3,7 and 14 after challenge, 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 STPS70c 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 STPS70c 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, and the efficacy of STPS70c was best.Experiment 9 Effects of two kinds of sPSs on expression of interIeukin-2 mRNA of chicken peripheral lymphocyte In order to study the action mechanism of two kinds of sPSs in immune-enhancing actions, the effect of STPS70c and sCPPSsoc on expression of IL-2 mRNA of chicken peripheral lymphocyte was determined taking non-modified TPStp as control. The chicken peripheral blood lymphocyte were cultivated in adding STPS70c, sCPPSsoc and TPStp at three concentrations. After cultivation of 12 h, the cells were collected and total RNA was extracted,The expression of IL-2 mRNA was determined by fluorescence quantitative RT-PCR assay. The results showed that STPS70c at 1.563 μg·mL-1 could promote the expression of IL-2 mRNA, which significantly stronger than PHA and TPStp. and there was a certain of dose-efficacy relationship. |
PDF Full Text Request