Antiviral And Immune-Enhancing Activities Of Sulfated Lycium Barbarum Polysaccharide And Sulfated Chinese Angelica Polysaccharide

Sulfate polysaccharide is a class of polysaccharides with sulfated group in its hydroxyl, including sulfate polysaccharides from plants, heparin, sulfated derivatives of natural neutral polysaccharides and synthesized sulfated polysaccharides. Many studies confirm that sulfate polysaccharide possesses stronger or new biological activities compared with natural polysaccharide. In order to obtain more sulfate polysaccharides, sulfated modificaton of natural polysaccharide can be performed. lycium barbarum polysaccharide (LBPS) and Chinese angelica polysaccharide (CAPS) are main effective ingredients of lycium barbarum L and Chinese angelica, respectively. In this research, LBPS and CAPS were selected as experimental materials and sulfatedly modified. The antiviral and immune-enhancing activities of sulfated LBPS (sLBPS) and sulfated CAPS (sCAPS) were determined through the tests in vitro and vivo, and further compared with sulfated astragalus polysaccharide (sAPS), sulfated epimedium polysaccharide (sEPS) and sulfated lentinan (sLNT) selected by previous researches of our laboratory. The purpose of this study is to validate the probability of sulfated modification to raise the bioactivity of LBPS and CAPS, probe into the dependablity of the bioactivity with the degree of sulfate group (DS) of sulfated polysaccharide, screen the best sulfated polysaccharide with antiviral and immune-enhancing activities and provide the materials for developing antivirus preparations and immunopotentiators from polysaccharides. The tests are divided into seven parts as follows:Experiment 1 Preparation of sLBPS and sCAPS Crude LBPS was extracted by-water decoction and ethanol precipitation and purified by Sevages’method to eliminate protein, permeate through G-75 Sephadex column and freeze drying, the purified LBPS were obtained. CAPS was provided by our laboratory. According to our previous established mathematical model on the degree of sulfate group (DS) of sulfated polysaccharide (sPS) related with modification condition, reaction temperature, reaction time and ratio of chlorosulfonic acid to pyridine, four modification conditions of each polysaccharide were designed, two kinds of polysaccharides were modified and four sLBPSs (sLBPS0.7, sLBPS1.1, sLBPS1.5, sLBPS1.9) and four sCAPSs (sCAPS0.6, sCAPS1.1, sCAPS1.9, sCAPS2.2) were obtained, respectively. They were taken as the materials of following experiments.Experiment 2 Effects of two kinds of sPSs on cellular infectivity of NDV in vitro The safe concentrations of four sLBPSs, four sCAPSs and non-sulfated LBPS and CAPS on chick embryo fibroblast (CEF) were determined by MTT assay. Every polysaccharide 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-and post-adding polysaccharide and simultaneous adding polysaccharide and virus after mixed. The changes of cellular infectivity (the A570 value and virus inhibitory rate) of NDV were compared. The result showed that during pre-adding polysaccharide, the A570 values of each polysaccharide at 1-5 concentration groups were significantly larger than that of corresponding virus control group except sLBPS0.7, LBPS and sCAPS0.6 group, the virus inhibitory rates of sLBPS1.1, sLBPS1.5, sLBPS1.9, sCAPS2.2and sCAPS1.1 group were larger than those of corresponding LBPS and CAPS group, respectively. During post-adding polysaccharide, the A570 values of each polysaccharide group at 1-5 concentration groups were significantly larger than that of corresponding virus control group except LBPS and sLBPSo 7 group, the virus inhibitory rates of sLBPSs except sLBPSo.7 were significantly larger than that of LBPS, and most of sCAPSs groups were larger than that of CAPS group. During simultaneous adding polysaccharides with virus after mixed, the A570 values of all the sulfated polysaccharide groups and CAPS group at 1-5 concentration groups were significantly larger than that of corresponding virus control group, the virus inhibitory rates of sLBPS1.5 and sLBPS1.1 group were significantly larger than that of LBPS group, and sCAPS2.2 and sC APS1.9 group larger than that of CAPS group. These results indicated that eight sulfated polysaccharides and CAPS at the suitable example-adding modes and doses could significantly inhibit NDV to infect CEF, sulfated modification could enhance the antiviral activity of LBPS and CAPS, which was in relation to the degree of sulfation (DS). In general evaluation the actions of sLBPS1.5 and sCAPS2.2 were stronger.Experiment 3 Effects of two kinds of sPSs on chicken peripheral lymphocyte proliferation in vitro Four sLBPSs, four sCAPSs and non-sulfated CAPS and LBPS at five concentrations were added into the cultured chicken peripheral blood lymphocyte in single or simultaneous with PHA, respectively. The changes of the lymphocyte proliferation (cellular A570 value and the lymphocyte proliferation rate) were determined by MTT method. The results showed that in single adding, the A570 values of sLBPS1.9, sLBPS1.5 and sLBPS1.1 at 5 concentration groups, sCAPS1.1, sCAPS1.9and sCAPS2.2 at 1-4 concentration groups were significantly larger than that of corresponding cells control group, the lymphocyte proliferation rates of sLBPS1.9, sLBPS1.5, sLBPS1.1,sCAPS2.2 and sCAPS1.9 group were significantly higher than those of LBPS and CAPS group, respectively. In simultaneous adding with PHA, the A570 values of all polysaccharides except sC APS0.6 group at 1-5 concentration groups were significantly larger than that of corresponding PHA control group, the lymphocyte proliferation rates of sCAPS2.2 and sCAPS 1.9 group were significantly higher than that of CAPS group. These results indicated that sLBPSs and sCAPSs at the suitable DS and doses could promote lymphocyte proliferation singly or synergistically with PHA, sulfated modification could enhance the immune-enhancing activity of LBPS and CAPS, in which there was a certain relativity to the DS of sulfated polysaccharide. In general evaluation the actions of sCAPS2.2, sCAPS1.9, s LBPS 1.9 and sLBPS1.5 were stronger.Experiment 4 Curative effect comparison of two kinds of sPSs on Newcastle disease in chickens sCAPS2.2and sLBPS1.5 with better antiviral effects were screened by the test in vitro. In this test the anti-NDV activities in vivo of them at high, middle and low three doses were further compared taking sulfated astragalus polysaccharide (sAPS), sulfated epimedium polysaccharide (sEPS) and sulfated lentinan(sLNT) selected by our previous experiments as control. Five hundred and fifty 21-day-old chickens were divided randomly into 11 equal groups and infected with NDV except blank control group. After the obvious symptoms appeared on alternate day, the chickens in six sPS groups were injected respectively with sCAPS2.2 and sLBPS1.5 at 4,2 and 1 mg dose, and three polysaccharide control groups, sAPS, sEPS and sLNT at 1 mg dose, respectively, once a day for three successive days. The clinical symptoms and death status were observed daily. On days 14 after challenge, mortality, cure rate and protective rate of every group were 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 of all polysaccharide groups except middle and low dose of sLBPS1.5, the mortalities were significantly lower and the protective rates were significantly higher than virus control group. In sAPS group, the mortality was lowest and the protection rate was highest, the cure rate of sCAPS2.2 at middle dose group was highest. The antibody titers of most polysaccharide groups were significantly higher than that of virus control group. These results indicated that several sulfated polysaccharides had certain therapeutic action for chicken Newcastle disease and could promote the antibody formation. The actions of sAPS, sCAPS2.2 at middle dose and sEPS were stronger.Experiment 5 Effects of the two kinds of sPSs on immune response of ND vaccine in chickens sCAPS2.2, sCAPS1.9. sLBPS1.9 and sLBPS1.5 with better immune-enhancing effects were screened by the test in vitro. In this test the immune-enhancing activities in vivo of four sPSs were further compared. One hundred and eighty 14-day-old chickens were divided randomly into 9 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 four sPS groups were injected with 2.0 mg of sCAPS2.2. sCAPS1.9, sLBPS1.9 and sLBPS1.5,in three polysaccharide control groups, with 2.0mg of CAPS, LBPS and crude CAPS (CAPSc), in immunization control and blank control 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 titers of sCAPS2.2 and sLBPS1.5 group at every time point, sCAPS1.9 and sLBPS1.9 group on days 14, 21 and 28 were significantly higher than that of corresponding immunization control group. The A570 value of sCAPS1.9, sLBPS1.9 and sLBPS1.5 group on days 14,21 and 28, sCAPS2.2 on days 7 and 21 were significantly larger than that of corresponding immunization control group. The lymphocyte proliferation rates of sLBPS1.9, sLBPS1.5 and sCAPS2.2 were significantly larger than that of corresponding immunization control groups. These results indicated that four sulfated polysaccharides could significantly promote the immune response of ND vaccine, whose effects were better than non-modified polysaccharides. In general evaluation the actions of sCAPS2.2 and sLBPS1.5 were stronger.Experiment 6 Effects of the two two kinds of sPSs on secretion of IFN-γ and IL-10 in chicken vaccinated with ND vaccine sCAPS2.2 and sLBPS1.5 with better immune-enhancing effects were screened by the test in vivo. In this test the effects of two kinds of sPSs on the secretion of IFN-γ and IL-10 in chicken vaccinated with ND vaccine were further compared. Three hundred and thirty 14-day-old chickens were divided randomly into 11 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 sPS groups were injected with 4,2,1 mg of sCAPS2.2 and sLBPS1.5, in three polysaccharide control groups, with 1.0 mg of sAPS, sEPS and sLNT, in immunization control and blank control 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. On days 28 after the first vaccination, the contents of serum IFN-y and IL-10 in SCAPS22 at middle and low dose, sAPS and sEPS group were determined. The result showed that the A570 values of sCAPS2.2 at low dose, sAPS and sEPS group at all time points, other polysaccharide groups at 1-3 time points were significantly higher than that of immunization control group. The lymphocyte proliferation rate of sAPS group was significantly higher while other polysaccharide groups were higher than that of immunization control group. The antibody titers of sCAPS2.2 at low dose, sAPS, sEPS and sLNT group at all time points while other polysaccharide groups at 2-3 time points were significantly higher than that of immunization control group. The IFN-y content of sCAPS2.2 at low dose and sEPS group were significantly higher than that of immunization control group, among which that of sCAPS2.2at low dose group was the highest. The IL-10 content of sCAPS2.2 at middle dose group was the lowest and the following was sEPS. These results indicated that these sulfated polysaccharides could significantly enhance the immune response of ND vaccine, promote the secretion of IFN-y. In general evalution the actions of sCAPS2.2 at low dose, sAPS and sEPS groups were stronger.Experiment 7 Effects of sCAPS on NP gene mRNA expression of NDV infected CEF sCAPS2.2 with better antiviral effect was screened by the test in vivo. In this test the effect of the sPS on NP gene mRNA expression of NDV infected CEF were further determined taking sAPS and sEPS as control. Chick embryo fibroblast (CEF) was cultivated, when CEF grew into monolayer, MEM was removed,2 mL of NDV was added into CEF plate, after incubated for 30 min at 4℃, virus solution was removed,2 mL of sCAPS2.2, sAPS and sEPS were added, respectively.The cells were collected after incubated for 72 h and total RNA was extracted. According to Primer Premier software, a pair of special RT-PCR primers of NDV was designed and 176 bp protein NP gene fragment was amplified, the relative expression quantity of NDV was determined by fluorescence quantitative real time RT-PCR assay. The results showed that the relative expression quantity of NP gene mRNA in every sulfated polysaccharide group was significantly lower than that of virus control group, in high dose of sAPS group was the lowest, and the following were low dose of sAPS and high dose of sCAPS2.2 group. These results indicated that sCAPS2.2, sAPS and sEPS could significantly inhibit NDV proliferation in CEF and the action of sAPS was stronger.