Yunpeng Fan

Lycium barbarum polysaccharide inhibits the infectivity of Newcastle disease virus to chicken embryo fibroblast

Lycium barbarum polysaccharide (LBPS) was extracted by water decoction and ethanol precipitation. After purification, four sulfated lycium barbarum polysaccharides (sLBPSs), sLBPS(0.7), sLBPS(1.1), sLBPS(1.5) and sLBPS(1.9), were prepared by chlorosulfonic acid-pyridine method respectively at four designed modification conditions. Four sLBPSs at 5 concentrations, within the safety concentration scope, and Newcastle disease virus (NDV) were added into cultivating system of chick embryo fibroblast (CEF) respectively in three modes, pre- and post-adding polysaccharide and simultaneous adding polysaccharide and virus after being mixed. The effects of sLBPSs on cellular infectivity of NDV were assayed by MTT method taking the non-modified LBPS as control. The results showed that sLBPS(1.5), sLBPS(1.9) and sLBPS(1.1) in three sample-adding modes, sLBPS(0.7) in simultaneous adding after being mixed could significantly inhibit the infectivity of NDV to CEF. The viral inhibitory rate of sLBPS(1.5) in pre- and simultaneous adding and sLBPS(1.9) in post-adding was the highest. Non-modified LBPS did not present significant effect in any sample-adding mode. These results indicated that sulfated modification could significantly enhance the antiviral activity of LBPS, which was correlated with the degree of sulfation (DS) of sLBPS. sLBPS(1.5) and sLBPS(1.9) possessed better activity and would be as the compositions of antiviral prescription.

Epimedium polysaccharide and propolis flavone can synergistically stimulate lymphocyte proliferation in vitro and enhance the immune responses to ND vaccine in chickens.

Four prescriptions, epimedium flavone plus propolis flavone (EF-PF), epimedium flavone plus propolis extracts (EF-PE), epimedium polysaccharide plus propolis flavone (EP-PF) and epimedium polysaccharide plus propolis extracts (EP-PE), were prepared and their immune-enhancing effects were compared. In test in vitro, the effects of them on chicken peripheral lymphocyte proliferation were determined by MTT method. The results showed that EP-PF group presented the highest stimulating index at most concentrations. In immune test, 300 14-day-old chickens were randomly divided into six groups and vaccinated with ND vaccine except for blank control (BC) group, re-challenged at 28 days of age. At the same time of the first vaccination, the chickens in four experimental groups were injected, respectively, with four prescriptions. The changes of the lymphocyte proliferation and antibody titer were determined. On day 28 after the first vaccination, the chickens except for BC group were challenged with NDV, the immune protective effect was observed. The results displayed that in EP-PF group, the antibody titers, lymphocyte proliferation and protective rate were the highest, the morbidity and mortality were the lowest. In dose test, 14-day-old chickens were randomly divided into five groups. The treatment and determinations were the same as the immune test except that the chickens in experimental groups were injected, respectively, with high, medium and low doses of EP-PF. The results revealed that in medium dose group, the antibody titers, lymphocyte proliferation and protective rate were the highest, the morbidity and mortality were the lowest. These results indicated that EP and PF possessed synergistically immune enhancement, EP-PF had the best efficacy, especially at medium dose, and would be expected to exploit into a new-type immunopotentiator.

In vitro antiviral activity of sulfated Auricularia auricula polysaccharides

Total Auricularia auricula polysaccharide (AAP(t)) was prepared by extracting and removing the proteins. Column chromatography was used to further graded it into AAP(1) and AAP(2). Three AAPs were modified by chlorosulfonic acid-pyridine method to obtain three sulfated AAPs (sAAPs), sAAP(t), sAAP(1) and sAAP(2), respectively. Three sAAPs and Newcastle disease virus (NDV) were added into cultivation system of chicken embryo fibroblast (CEF) in three manners, pre-, post- and simultaneous-adding polysaccharide with NDV respectively, taking three non-modified AAPs as control. Their anti-viral activities were compared by MTT method. The results showed that sAAPs and AAPs at a certain concentration could significantly inhibit the cellular infectivity of NDV in three manners. The effects of sAAPs were better than that of AAPs. It indicated that sulfated modification could enhance the antiviral activity of AAP. sAAP(1) and sAAP(t) possessed stronger activity and would be as the component of a new-type antiviral drug.

The immunological activity of propolis flavonoids liposome on the immune response against ND vaccine.

Three hundred and fifty 14-day-old chickens were randomly assigned to 7 groups. At the same time of vaccination with Newcastle disease vaccine, the chickens in experimental groups were injected with propolis flavonoids liposome (PFL) at three doses, PF and blank liposome, respectively. The titer of serum antibody, concentrations of immunoglobulins G (IgG) and immunoglobulins M (IgM), activity of lymphocytes proliferation and concentrations of cytokines were measured. The results showed that three doses of PFL could significantly enhance antibody titer, concentrations of IgG, IgM, and promote lymphocyte proliferation, interferon-γ and interleukin-2 secretion, and its high and medium doses possessed the best efficacy. In general evaluation, the efficacy of PFL was the best, with certain of dose- and time-effect relationships. These findings indicated that the immunological activity of PF could be enhanced with liposome encapsulation.

Optimization on condition of glycyrrhetinic acid liposome by RSM and the research of its immunological activity.

The aim of this study is to prepare glycyrrhetinic acid liposome (GAL) and optimize the preparation condition and to investigate further whether liposome could promote the immunological activity of glycyrrhetinic acid (GA). GAL was prepared using a film-dispersion method and the preparation conditions of GAL were optimized with response surface methodology (RSM). Moreover, GAL prepared under the optimal preparation conditions was added into chickens T and B lymphocytes in vitro. The optimal preparation conditions for GAL by response surface methodology was as follows: ratio 9:1, soybean phospholipid cholesterol (w/w) 2.5:1 and water bath temperature 31 °C. Under these conditions, the experimental encapsulation efficiency of GAL was 83.46 ± 0.55%, which was close with the predicted value. Therefore, the optimized preparation condition is very reliable. The results showed that GAL could significantly promote T and B lymphocytes proliferation singly or synergistically with PHA and LPS and the concentration of immunoglobulins G (IgG) and immunoglobulins M (IgM). These results indicated that liposome could significantly improve the immunological activity of GA and drug action of GA. GAL demonstrates the significant immunological activity, which provides the theoretical basis for the further experiment in vivo.

Optimization of sulfated modification conditions of tremella polysaccharide and effects of modifiers on cellular infectivity of NDV

Based on our previous research, sulfated modification conditions of Tremella polysaccharide (TPS), the chlorosulfonic acid to pyridine (CSA-Pry) ratio, reaction temperature and time, were optimized by L(9) (3(4)) orthogonal design taking the yield and degree of sulfation (DS) of modifiers as indexes. Two TPSs, TPS(tp) and TPS(70c), were modified under optimized conditions. The effects of two modifiers, sTPS(tp) and sTPS(70c), on cellular infectivity of NDV were determined by MTT method taking the non-modified TPS(tp), TPS(tc) and TPS(70c) as controls. The results showed that the optimized modification conditions were reaction temperature of 80°C, CSA-Pry ratio of 1:6 and reaction time of 1.5h. Five polysaccharides at proper concentrations could significantly inhibit the infectivity of NDV to CEF. The virus inhibitory rates of sTPS(tp) at 1.563 μg mL(-1) group were the highest and significantly higher than those of other three non-modified polysaccharide groups in three sample-adding modes. This indicated that sulfated modification could significantly improve the antiviral activity of TPS. sTPS(tp) possessed the best efficacy and would be as a component of antiviral polysaccharide drug.

Astragalus polysaccharide and oxymatrine can synergistically improve the immune efficacy of Newcastle disease vaccine in chicken.

Three hundred and sixty 14-day-old chickens were divided into seven groups. The chickens, except for blank control group, were vaccinated with Newcastle disease vaccine, repeated at 28 days old. At the same time of the first vaccination, the chickens in three astragalus polysaccharide-oxymatrine (AP-OM) groups were orally administrated respectively with the mixture of AP-OM at high, medium and low concentrations, in astragalus polysaccharide (AP) group and oxymatrine (OM) group, with corresponding medicine, in non-medicine (NM) control group, with equal volume of physiological saline, once a day for 3 successive days. On 14, 21, 28, 35 and 42 days after the first vaccination, the changes of peripheral lymphocyte proliferation and serum antibody titers of the chickens were determined by MTT method and hemagglutination inhibition test. On 14, 28 and 42 days after the first vaccination, the serum IL-2 concentration was determined by Enzyme-linked Immunosorbent Assay (ELISA). The results showed that at most time points, the lymphocyte proliferation, antibody titers and IL-2 concentrations of 5 medicine-administrating groups were significantly higher than that of corresponding NM group. At some time points, the lymphocyte proliferation, antibody titers and IL-2 concentrations in high and medium doses of AP-OM groups were significantly or numberly higher than those in AP group and OM group. It indicated that AP-OM could significantly improve the immune efficacy of Newcastle disease vaccine, astragalus polysaccharide and oxymatrine possessed synergistical immunoenhancement.

Effect of epimedium polysaccharide-propolis flavone immunopotentiator on immunosuppression induced by cyclophosphamide in chickens.

Two hundred and fifty 11-day-old chickens were randomly assigned into 5 groups and except normal control group injected with cyclophosphamide once a day for 3 successive days. At day-14-old, all chickens were vaccinated with Newcastle disease vaccine. At the same time of the first vaccination, the chickens in three experimental groups were injected respectively with epimedium polysaccharide-propolis flavone immunopotentiator (EPI) at three dosages, once a day for 3 successive days. On days 7, 14, 21 and 28 after the first vaccination, the serum antibody titer and IgG, IgM, IFN-γ and IL-6 concentrations, peripheral lymphocyte proliferation, including immune organ index on day 28, were measured. The results demonstrated that EPI at high and medium doses could significantly enhance antibody titer and IgG, IgM, IFN-γ and IL-6 concentrations, promote lymphocyte proliferation and enlarge immune organ index as compared with model control group. This indicated that EPI could effectively resist the immunosuppression.

Immuno-enhancing activity of sulfated Auricularia auricula polysaccharides

The crude total Auricularia auricula polysaccharide (AAPct) was extracted by water decoction and ethanol precipitation, protein was removed to obtain total A. auricula polysaccharide (AAPt), then was graded into AAP1 and AAP2 through column chromatography. sAAPt, sAAP1 and sAAP2 were prepared by chlorosulfonic acid-pyridine method. In vitro test, the effects of sAAPt, sAAP1, sAAP2, AAPt, AAP1 and AAP2, on chicken peripheral lymphocytes proliferation were compared. The results showed that sAAPt and sAAP1 demonstrated better effect. In vivo test, 14-day-old chickens were injected respectively with sAAPt, sAAP1, AAPt and AAP1 at the first vaccination of ND vaccine, once a day for three days. On days 7, 14, 21 and 28 after the first vaccination, the peripheral lymphocytes proliferation and antibody titer were determined. The results indicated that sAAPt possessed the best efficacy and would be expected to be used as a component of a new-type immunopotentiator.

Immunological adjuvant efficacy of glycyrrhetinic acid liposome against Newcastle disease vaccine

The aim of this study is to investigate whether the activity of inducing immune response of glycyrrhetinic acid (GA) could be enhanced after GA was encapsulated with liposome. Three hundred and fifty 14-day-old chickens were randomly assigned to 7 groups and vaccinated with Newcastle disease (ND) vaccine. Simultaneously, the chickens in experimental groups were injected with the glycyrrhetinic acid liposome (GAL) at three doses, GA and blank liposome, respectively. The activity of serum antibody titer, concentrations of immunoglobulins G (IgG) and immunoglobulins M (IgM), lymphocytes proliferation, the proportions of lymphocyte subpopulations (CD4(+) and CD8(+)) was determined after vaccination. GAL was evaluated for inducing humoral immunity and cellular immunity in chicken against Newcastle disease (ND) vaccine. The results showed that GAL not only could significantly enhance the antibody titers, IgG and IgM in ND vaccine immunized chicken, but also significantly promote the lymphocyte proliferation and the proportions of CD4(+) and CD8(+), as comparison with GA, BL and VC control groups. Moreover, the effects appear a dose-dependent manner and a time-dependent manner. These indicated that GAL could significantly promote the activation potential of humoral immunity and cellular immunity in chicken and present certain dose-effect and time-effect relationships. The formulations of GA and liposome can further enhance the immune response against ND vaccine compared with the adjuvant alone.