Study On Preparation And Immunological Adjuvanticity Of Propolis Flavonoids Liposome

Liposome is a synthetic bilayer membrane vesicle with phosphorus. Liposome can wrap in water-soluble drugs and fat-soluble drugs for their hydrophilic and hydrophobic. Liposome has intrinsic ability to extravasate into tissues with increased vascular permeability, slow release drug, because it’s membrane possesses a compatibility with the cell membrane. In addition, the liposome can reduce toxicity and side effect of drugs and impove their high stability in the blood. Many researches found that, through lots of the traditional Chinese herbal medicine or traditional Chinese herbal medicine ingridients have strong biological activity, for example propolis flavonoids were reported that it, as adjuvant, possessed immune enhancement, their not solubility in water, or quick metabolism in vivo result in not sustain longer drug action. These drugs are encapsulated with liposome, not only can solve the above problems, but also can be play the advantagements of drugs and liposome. Base on all above mentioned, this study is to prepare the PFL by ethanol injection method and the optimal prescription for preparation of PFL was selected with response surface methodology; and the immunological adjuvant activity of PFL was researched including the activity of peripheral T lymphocyte proliferation, antibody titers, the concentrations of immunoglobulin and cytokines. The aim of this study is to develop high-effect and low-toxicity newtype of immunological adjuvant. The details were divided into five parts as follows:Experiment 1 Preparation condition optimization of propolis flavone liposome by response surface methodology To optimize the preparation conditions of propolis flavone (PF) liposome. Propolis flavone (PF) liposome was prepared by ethanol injection method. The supercritical preparation conditions of PF liposome was optimized with the conditions of lecithin to drug ratio, lecithin to cholesterol ratio, and the speed of injection by response surface methodology based on single factor experiments. According to Box-Benhnken experimental design, the response surface analytical method with 3 factors and 3 levels was used to optimize the preparation conditions of PF liposome. The result showed that the optimal preparation conditions were as follows:the ratio of lecithin to drug was 9.6:1, the ratio of lecithin to cholesterol was 8.5:1, the speed of injection was 0.8 mL·min-1. The PF liposome prepared under the optimized conditions has high encapsulation efficiency. The entrapment rate of propolis flavone liposome measured by validated test was 91.67% with relative error of 0.086% to model prediction, and had a good reproduction quality.Experiment 2 Effect of PFL on lymphocyte proliferation in chickens in vitro The effects of PFL and PF on growth and proliferation of the peripheral lymphocyte were compared by MTT method. PFL and PF were diluted to eleven concentrations with RPMI-1640. The peripheral lymphocyte in 96-well plates were exposed to drugs at series of concentrations, four wells each concentration, to be observed the results after 48 h. In order to study PFL and PF on their effect of the peripheral lymphocyte proliferation, at five concentrations were added into the peripheral lymphocyte in vitro, and cultured 48 h. The peripheral lymphocyte proliferation was determined with MTT method. These results showed that PFL was safety in all of concentrations, PF and BL maximal safety concentrations falled down; all of PFL and PF could promote the lymphocyte proliferation, at 60-3.75μg·mL-1, PFL could stimulate the lymphocyte proliferation singly or synergistically with PHA both could promote the lymphocyte proliferation, at the same time, at 60-15μg·mL-1, PFL singly or synergistically with PHA could stimulate the lymphocyte proliferation which had better effect than PF.Experiment 3 Effects of PFL on mRNA Expression of cytokine in chickens in vitro In order to study the effect of PFL on the mRNA expression of cytokines in vivo, The chicken peripheral lymphocyte were cultivated in adding PFL, PF and BL at three concentrations respectively. After cultivation of 36 h, the cells were harvested and total RNA was extracted. The mRNA expression of IL-2, IL-4 and IFN-γ was evaluated by fluorescence quantitative RT-PCR assay. The results showed that PFL and PF all could improve the expression level of IL-2、IL-4 and IFN-γ mRNA, and the action of PFL were significantly strong than PF at 60 μg·mL-1,30μg·mL-1 and 15 μg·mL-1. At 60μg·mL-1, the action of PFL was the strongest, at 30μg·mL-1, the action of PFL was stronger, with certain of dose-effect relationships. It might be one of the immune enhancement mechanisms that PFL could promote the secretion of IL-2、IL-4 and IFN-y.Experiment 4 Effect of PFL on immune response of ND vaccine in chickens In order to compare the immune-enhancing actions of PFL and PF in vivo, the effects of PFL and PF on immune response of ND vaccine in chickens were determined. Two hundred and ten 14-day-old chickens were randomly divided into seven groups and vaccinated with ND vaccine except for blank control group, repeated vaccination at the age of 28 days. At the same time of the first vaccination, the chickens in five experimental groups were intramuscularly injected with 0.5 mL of PFL at high, medium and low dose, PF and BL, respectively, in vaccine control and blank control groups,0.5 mL of physiological saline for three days. On days 7,14,21,28,35 and 42 after the first vaccination, the blood was sampled for determination of lymphocytes proliferation by MTT method, serum titer of hemagglutination inhibition (HI) antibody by micro-method. The results showed that, PFL significantly promoted T lymphocyte proliferation and raised antibody titer, as comparision with PF, BL and VC groups. Moreover, the effects appear a dose-dependent manner and a time-dependent manner. These findings indicated that the immunological adjuvant activity of PF could be enhanced with liposome encapsulation and its high and medium dose possessed the best efficacy.Experiment 5 Effects of PFL on immunoglobulin and cytokine levels of serum in chickens In order to further study the immunological adjuvant activity of PFL. Two hundred and ten 14-day-old chickens were randomly divided into seven groups and vaccinated with ND vaccine except for blank control group, repeated vaccination at the age of 28 days. At the same time of the first vaccination, the chickens in five experimental groups were intramuscularly injected with 0.5 mL of PFL at high, medium and low dose, PF and BL, respectively, in vaccine control and blank control groups,0.5 mL of physiological saline for three days. On days 7,14,21, 28,35 and 42 after the first vaccination, the blood was sampled for determination of the concentrations of interferon-y (IFN-y), interleukin-2 (IL-2), IgG (Immunoglobulin G), IgM (Immunoglobulin M) with enzyme-linked immunosorbent assay (ELISA). The results showed that three doses of PFL could significantly enhance concentrations of IgG, IgM and promote interferon-y and interleukin-2 secretion, with certain of dose -and time-effect relationships, and its high and medium doses possessed the best efficacy. These findings indicated that the immunological activity of PF could be enhanced with liposome encapsulation.