| Polysaccharides had many kinds of biological activities, especial better antiviral action and anticancer action, which demonstrated a wide perspective in domain of pharmacectic. These activities of polysaccharides could be enhanced by molecular modification, and some polysaccharides which had non-activities or small activities could abtain larger activities. Sulfated polysaccharide is a kind of polysaccharides containing sulfate ions. More sulfated polysaccharides can be obtained by using sulfating methods. Lentinan was selected as object and its extraction, isolation, purification, modification by chlrosulfonic acid-pyridine to prepare three sulfated polysaccharides and some biological activities were studied compared with lentinan in this research. In order to comparing its activities with that of others four sulfated polysaccharides (sAPS60, sEPS5, sCAPS11 and sLBP14 selected before in our lab), the anti-virus and enhancing immunity activities of five sulfated polysaccharides was determined. The details are divided into nine parts as follows:Test 1 The extraction and purification of Lentinan Lentinan, which had the ability of anti-virus and immunoenhancement according to literatures, was selected as objects in the experiment. Lentinan was crudely extracte by ultrasonc-water- extracting-precipitation method and removed protein from polysaccharide by the Sevag’s method, removed pigment by H2O2. Then, the polydsaccharide was refined by gel filtration with Sephadex G-200. The lentinan contents were assayed by vitriol-phenol method, protein contents by the Bradford G-250 methed. The results showed that the extractive rate, net content and protein content of LNTc were 8.4%,30% and 65% respectively. With the progress of wiping off protein the yield of polysaccharide and content of protein reduced gradually, but the content of polysaccharide elevated little by little. And the content of LNTp was heightened to 91.0% and protein was eluted synchronously to 5.3% after removing protein. Test 2 Sulfated modification of lentinan Sulfation of lentinan was prepared by chlrosulfonic acid-pyridine to prepare the material of the following tests. Reagent proportion, reaction temperature and reaction time were optimized with yield of sLNT and substitution degree of SO42+(DS) as index. The results showed that the optimum reaction condition as following:the reaction temperature was 60℃, the reaction time was 4 h and the ratio of chlrosulfonic acid and pyridine was 1:2～1:6. Then three sLNTs were sulfated at optimal reaction temperature and time with ratio of chlrosulfonic acid (1:2,1:4 and 1:6) and pyridine. And the DS of sLNT1, sLNT2 and sLNT3 were 1.37,0.98 and 0.69 and the recycle ratio of theirs were 62.5%,60.5% and 63.5%.Test 3 Effect of sulfated LNT on cellular infectivity of NDV, IBDV and IBV In order to comparing the effect of three sLNTs on cellular infectivity of NDV (Newcastle disease virus, NDV), IBDV (Infectious bursal disease virus, IBDV) and IBV (Infectious bronchitis virus, IBV) to Chicken embryo fibroblast (Chicken embryo fibroblast, CEF) in vitro. First of all, The safe concentration of four polysaccharides of LNT on growth of CEF were compared by MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) method. Then four polysaccharides of LNT were diluted to five concentrations with MM in of all their safe concentration and added into CEF monolayers by three models, pre-adding polysaccharide, post-adding polysaccharide and simultaneous adding polysaccharides and bursal disease virus (NDV, IBDV and IBV) by mothed of MTT to observe the cellular infectivity. These results showed that the antiviral activity of LNT was promoted after sulfation and had relation to their concentrations, That was the sLNTs had effect in concentration of 0.4μg·mL-1～5.9μg·mL-1 but LNT only in 1.5μg·mL-1～5.9μg·mL-1,and the effect of high concentration was better then its low. And the models of pre-adding polysaccharide was better than other two models on antiviral activity and the effect of antiviral activity of sLNT2 was best in three sulfated LNTs.Test 4 Effects of the sulfated lentinan on cultivated lymphocyte proliferation In order to screening the optimum sulfated modification conditions of lentinan with stronger immunoenhancement action in vitro, The effects of the sulfated lentinans on cultivated lymphocyte proliferation was determined in this test. The three sLNT and LNT at five doses were added in the peripheral blood or spleen lymphocyte, singly or synergistically with irritant agent, and cultured 48 h. The peripheral blood lymphocyte proliferation was determined with MTT method. The results showed that the sLNTs had effect in concentration of 0.4μg·mL-1～5.9μg·mL-1 but LNT only in 1.5μg·mL～1-5.9μg·mL-1, and the effect of sLNTs was better then that of LNT, which indicated that the effect of stimulating lymphocyte proliferation of LNT could be enhanced by sulfated modification; And the effect of stimulating lymphocyte proliferation of sLNTs had relation to their concentrations(μg·mL-1), that was 5.9>3.0>1.5>0.8>0.4. And the A570 of sLNT2 was the biggest in that of three sLNTs, which indicated.that the effect of stimulating lymphocyte proliferation activity of sLNT2 was the strongest in that of three sLNTs.Test 5 Effect of sulfated lentinan on immune effect of New castle disease vaccine In order to validating the effect of three sLNTs on immunoenhancement to ND vaccine. In immune response test, four hundred and fifty 14-day-old chickens were randomly divided into 9 groups and vaccinated with New castle disease vaccine, repeated vaccination at 28-day-old. At the same time of the first vaccination, the chickens in 6 sLNT groups were intramuscularly injected respectively with sLNT1, sLNT2 and sLNT3 at high and low dose, in 2 lentinan (LNT) control groups, LNT. Before vaccination and in days 6 weeks after vaccination, the dynamic change of serum HI antibody and peripheral T lymphocyte proliferation was determined respectively. In immune protective test, three hundred and fifty 14-day-old chickens were divided randomly into 7 groups and vaccinated with New castle disease vaccine except for non-vaccinated (NV) group. At the same time, the chickens in 3 sLNT groups were intramuscularly injected respectively with sLNT1, sLNT2 and sLNT3 0.5 mL at 8.0 mg·mL-1, in LNT control group, LNT, and in the other groups, with 0.5 mL physiological saline. On day 21 after vaccination, except for NA2 (non-adjuvant, NA) group, the chickens were attacked with intramuscular injection of New castle disease virus (NDV). Before attack and on days 3,6,9,12 and 15 after attack, the dynamic change of serum HI antibody and peripheral T lymphocyte proliferation was measured. The results showed that three modified sLNTs could significantly enhance antibody titer and promote lymphocyte proliferation as compared with non-modified LNT. In enhancing antibody titer, effects of their high doses were better, in promoting lymphocyte proliferation, low dose in immune response test. In immune protective test, three sLNTs could significantly reduce morbidity and mortality and increase protection rate of chickens attacked with NDV, and accelerate the antibody production and lymphocyte proliferation, which were better than that of non-modified LNT. In general evaluation, the effect of sLNT2 was best among sLNTs. Test 6 Effect of sulfated lentinan on immune response of IBD In order to inspecting the effect of three sLNTs on immunoenhancement to IBD vaccine. Four hundred and fifty 14-day-old chickens were divided randomly into 9 groups. The chickens in eight experimental groups were intramuscularly injected respectively with three sulfated lentinans (sLNTs), sLNT1, sLNT2 and sLNT3 and LNT, at high and low dose, in free adjuvant control group, physiological saline, after 4 days inoculated with IBD vaccine and the second vaccination after 14 days. Before vaccination and on days 7,14,21,28 and 35 after the first vaccination, the blood samples were collected from brachial vein for determination of serum antibody and the blood samples were collected by cardiopuncture for determination of peripheral T lymphocyte proliferation. And spleen, bursa and thymus were weighed randomly from each group for calculation of immune organ index. The results showed that three modified sLNTs could significantly enhance antibody titer, promote lymphocyte proliferation and the immune organ index markedly. At high dose, their effects of enhancing antibody titer were the better, at low dose, promoting lymphocyte proliferation, which were better than those of non-modified LNT and presented the relations to dose- and time-effect. In general evaluation the efficacy of sLNT2 was best.Test 7 The comparison between the five sulfated polysaccharide on resistance and elimination of NDV to infect CEF In order to screening the better antiviral activity of sulfated polysaccharides in vitro, the compare between the five sulfated polysaccharide on resistance and elimination of NDV to infect CEF was determined. In this paper, the maximal safe concentrations of sLNT2 and other four sulfated polysaccharides (sAPS60, sEPS5, sCAPS11 and sLBP14) selected befour in our lab to CEF were determined at first. And then the antiviral activity of the five sulfated polysaccharides against NDV on CEF was compared by MTT assay. The concentration of NDV in foster system of CEF was determined by fluorescence quantitative PCR assay. The results showed that the sEPS5, sAPS60 and sLNT2 had higher abilities on inhibitting cellular infectivity of NDV in five sulfated polysaccharides. And the order of inhibitting cellular infectivity activity of the five sulfated polysaccharides was sEPS5>sAPS60>sLNT2>sLBP14, sCAPS11, and sEPS5, sAPS60 and sLNT2 had the better activity of inhibitting cellular infectivity effect.Test 8 The comparison between the five sulfated polysaccharides on lymphocyte proliferation, secreting IL-2 and IL-6 In order to screening better immunoenhancement of the sulfated polysaccharides on in vitro, the compare between the five sulfated polysaccharide on immunoenhancement was determined. First of all, the safe concentration of the five sulfated polysaccharides was determined. Then the five sulfated polysaccharides were added in the peripheral lymphocyte, singly or synergistically with ConA. The peripheral lymphocyte proliferation was determined with MTT method and the contant of IL-2 and IL-6 in lymphocyte secretion were compared by method of ELISA (enzyme linked immunosrodent assay, ELISA). The results showed that all of the five sulfated polysaccharides could stimulate the lymphocyte proliferation singly or synergistically with ConA. The order of stimulating the peripheral lymphocyte proliferation was sAPS6o> sEPS5>sCAPS11>sLBP14 and sLNT2. Furthermore, the stimulating the lymphocyte proliferation by polysaccharides had compact relation to the concentration of polysaccharides. And five sulfated polysaccharides could stimulate the lymphocyte proliferation synergistically with ConA to secrete IL-2 and IL-6, which activity sAPS60 and sEPS5 had better.Test 9 The comparison between the three sulfated polysaccharides on instrengthening the effect of ND vaccine In order to screening the optimum sulfated polysaccharide with stronger immunoenhancement further in vivo, one hundred and seventy-five 14-day-old chickens were randomly divided into 5 groups and vaccinated with New castle disease vaccine, repeated vaccination at 28-day-old. At the same time of the first vaccination, the chickens in 3 sulfated polysaccharides were intramuscularly injected respectively with sLNT2, sAPS60 and sEPS5 0.5 mL-1. At the same time and on day after vaccination 7,14,21, the blood samples were collected from brachial vein for determination of serum antibody and on day after vaccination 14 and 21, the blood samples were collected by cardiopuncture for determination of peripheral T lymphocyte proliferation and secreting IL-2 and IL-6. On day after first vaccination 23, all chickens were challenged NDV except the negative group, and morbidity, mortality, protective rate were statistics. The results showed that that the immunoenhancement of adjuvant groups were better than those of non- adjuvant. Among of adjuvant, sAPS60 and sEPS5 had better effects, and then was sLNT2. The sulfated polysaccharide adjuvants were optimal in the effect of promoting serum ND-HI antibody titer, T lymphocyte proliferation, and secreting IL-2 and IL-6 of T lymphocyte, increasing protective rate. |
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