| The virus infectious diseases are always the most serious diseases to harm domestic animal and poultry industry. Up to now there are no effective therapeutic methods and vaccination is the most commonly used preventive measure. Because of the appearance of some variant strain and velogenic strain, the immunity effect is not satisfied. In order to gain better effect the combination of immunopotentiators is necessary. Due to the disadvantages of the extant immunopotentiators, it becomes so urgent to study and develop a new-type immunopotentiators with high efficacy and low toxicity. Many researches indicated that many Chinese herbal medicines have better immunoenhancement and the immunopotentiators made with traditional Chinese medicine possessed remarkable advantage. This study is based on our previous researches, epimedium polysaccharide (EPS), epimedium flavone (EF), epimedium extract (EE), propolis flavone (PF) and propolis extract (PE) were chosen and composed into prescriptions. Their effective immunoenhancements were compared to select a better prescription named epimedium propolis flavone immunopotentiator (EPI). Then, some studies were carried out including the dosage, dosage form, preparation technology, quality control, safety, clinical potency and action mechanism. The details are divides into nine parts as follows:Experiment I. The screening test of prescription Five prescriptions, epimedium polysaccharide plus propolis flavone (EPS-PF), epimedium flavone plus propolis flavone (EF-PF), epimedium polysaccharide plus propolis extract (EPS-PE), epimedium flavone plus propolis extract (EF-PE) and epimedium extract plus propolis flavone (EE-PF) were prepared. In vitro test, the effect of EPS-PF, EF-PF, EPS-PE and EE-PF on chicken peripheral lymphocyte proliferation was determined by MTT method. The results showed that EPS-PF had the best effect. In vivo test, four hundred and twenty 14-day-old chickens were randomly divided into 6 groups and vaccinated with ND vaccine except for blank control (BC) group, rechallenged at 28 days of age. At the same time of the first vaccination the chickens in four experimental groups were injected respectively with 0.5 mL of four prescriptions (EPS-PF, EF-PF, EPS-PE and EE-PF), in vaccine control (VC) and BC groups,0.5 mL of physiological saline. On days 7,14,21 and 28 after the first vaccination, blood was sampled for determination of lymphocyte proliferation and serum hemagglutination inhibition (HI) antibody titer. The indexes of immune organs were also measured. The results displayed that in EPS-PF group, the antibody titer and lymphocyte proliferation were significantly higher than other groups, and the most indexes of immune organs were significantly higher than other groups on days 14-28 after the first vaccination. In optimazing test for prescription, the effect of EPS-PF and EE-PF on serum antibody titers in chickens vaccinated with ND vaccine was compared. The results showed that the immune effect of two prescriptions were similar. Therefore, EE and PF were determined as the constituents of compound Chinese herbal medicinal flavone immunopotentiator, and named epimedium extract-propolis flavone immunopotentiator (EPI).Experiment II. The screening test of EPI dosage Compared the immunoenhancement effect of EPI in different dosages. In preliminary screening test, EPI was diluted into 7 dosages.14-day-old chickens were vaccinated chickens with ND vaccine, rechallenged at 28 days of age. At the same time of the first vaccination, the chickens in seven experimental groups were intramuscularly injected respectively with 1.5、1.25、1.0、 0.75、0.5、.25、0.125 mg of EPI, in VC and BC groups,0.5 mL of physiological saline. On days 7,14,21 and 28 after the first vaccination, blood was sampled for determination of serum hemagglutination inhibition (HI) antibody titer, and the reaction of chickens after administration was observed. The result showed that the antibody titers in each dosage group were numberly or significantly higher than VC and BC groups, the dosage of 0.75 mg had the best efficacy, and the chickens in each group had no any untoward reaction after administration. In confirmatory test, the optimal dosage of 0.75 mg screened by preliminary screening test was regard as medium dosage, and then the high dosge (1.25 mg) and low dosage (0.25 mg) were selected out according to arithmetic progression. Then, immunologic test was repeated. On days 7,14,21 and 28 after the first vaccination, blood was sampled for determination of lymphocyte proliferation and serum hemagglutination inhibition (HI) antibody titer. The result showed that the lymphocyte proliferation and antibody titer in three dosage groups were numberly or significantly higher than VC and BC groups at four time points, and the medium dosage (0.75 mg) possessed the best efficacy. Therefore,0.75 mg of EPI for each chicken was determined as the optimal dosage for clinical applicationExperiment Ⅲ. The screening test of dosage form of EPI EPI was made into three dosage forms in this experiment, as following:liposome, suspension and watery solution. Their immunoenhancement effect was compared. In test in vitro, the three dosage forms of EPI 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 highest lymphocyte proliferation rate) was determined by MTT method. The results showed that the three dosage forms of EPI could significantly promote T and B lymphocytes proliferation singly or synergistically with PHA and LPS at most concentrations. In test in vivo, two hundred and fifty 14-day-old chickens were randomly divided into 5 groups and vaccinated with ND vaccine. At the same time of the first vaccination, the chickens in three dosage form groups were injected with 0.5 mL of the three dosage forms of EPI respectively. On days 7,14,21 and 28 after the first vaccination, the serum antibody titer, content of IFN-y and IL-6 and peripheral lymphocyte proliferation were determined. The results showed that all the three dosage forms could promote lymphocyte proliferation, enhance antibody titer, and promote the secretion of IFN-y and IL-6. Above all, after comprehensive evaluating, the liposome had the best immunoenhancement effect, suspension was followed. Because the price of liposome was higher, and the stability was less than suspension, finally, suspension was choosed as the dosage form of EPI.Experiment Ⅳ. Study on the preparation technology of EPI The extract of EE and PF, and the content of suspending agent were studied. In EE extraction test, the extract conditions of EE, three decoction time and adding water multiple, were optimized by L9(34) orthogonal test taking the yield of epimedium extract and the content of epimedium polysaccharide as index. The results show that of the effects of adding water multiple on yield and content were greatest. The optimum extract technology was the ratio of water to epimedium of 20:1, decoction 2 times, the first time of 1.5 hours, the second of 1 hour. In PF extraction test, we compared the differences between ethanol dissolved method and ethanol dissolved-acetidin extraction method, taking the yield, content and net content of PF as the index. The results showed that there were no significant differences for three indexes of PF through two methods extraction, but the ethanol dissolved method was more economic and convenient, so the ethanol dissolved method was selected for extracting PF. In screening test for the contentcontent of suspending agent, based on preliminary test, glycerin and polyethylene glycol (PEG)-400 were screened out as suspending agent. Then seven concentrations of the solvent of PF (alcohol) and two kinds of suspending agents were compared for the effect on the solubility of PF, the characteristics and stability of preparation. Finally, the contents of them in EPI were determined that the alcohol, glycerin and PEG-400 were 6.0%,3.5% and 6.5% respectively.Experiment Ⅴ. Study on quality control of EPI The identity and content assay of EPI were studied. In icariin identity test, the chromatogram effects of three kinds of samping volume and two kinds of application of sample volume were compared. The results showed that the deflniting power and depth of spot were increased along with the increase of samping volume and application of sample volume. Finally,10 ml and 4μl were chosen as the optimal samping volume and application of sample volume respectively. In norizalpinin and chrysin identity test, the chromatogram effects of four kinds of developing solvent and five kinds of application of sample volume were compared. The results showed that the sample had the best separation effect when the developing solvent was methylbenzene-acetidin-formic acid (10:4:3). The depth of spot was increased along with the increase of application of sample volume, and the tail formation phenomenon also became aggravation gradually. Therefore,3μl was chosen as the optimal application of sample volume. In content assay test, high performance liquid chromatography was established for assaying the content of icariin, norizalpinin and chrysin in EPI, and the effectiveness of this method was proved. The results indicated that the reference substance of icariin had better linear relationship in 2.5-80 μg/ml, norizalpinin and chrysin in 15.625-250μg/ml, the established method possessed higher degree of precision and coefficient of recovery, and the sample solutions kept stable in nine hours.Experiment Ⅵ. Determination on safety of EPI According to the methods stipulated by the experiment technical literature of veterinary drugs, the acute toxicity, long-term toxicity and special toxicity of EPI were determined. The results of acute toxicity test showed that the mice had some untoward reactions half an hour after administration, such as poor spirit, anorexia and so on, but they restored to normal quickly, and then did not appear any paradoxical reaction again. There was no death in all the mice within 14 days, and the maximum dosage was 6000 mg/kg. The results of long-term toxicity test showed that during the administration period, the body weight of mice in high dose group was decreased, but there were no significant differences between them. The total white cell count of high dose group was significantly larger than control group. There were no significant differences in ALT, AST, ALP and UN among all groups. The spleen index of high and medium dose groups was significantly higher than control group, and there was no obvious abnormal change in autopsy and histology examination. All above indices returned to normal on days 14 after drug withdrawal. The results of special toxicity test showed that EPI had no sensitization in vivo, no hemolyzation in vitro and no stimulatory for the quadriceps femoris muscle and eye tunica conjunctiva of rabbit. These results indicated that EPI had no toxicity, long-term toxicity, and special toxicity. Therefore, the clinical application of EPI was safe.Experiment Ⅶ. Determination of adjuvanticity of EPI The immunoenhancement effect of EPI was determined on avian influenza (AI) vaccine and Newcastle disease (ND) vaccine. In AI immunologic test, AI vaccines were made into high, medium and low dose EPI adjuvant, oil adjuvant (OA) and non-adjuvant (NA) vaccines contained equivalent AI antigens with inactivated antigens, OA materials and water for injection. Three hundred 14-day-old chickens were randomly divided into 6 equal groups and hypodermicly injected with three AI vaccines contained EPI adjuvant (high, medium and low dose), and then taking oil adjuvant (OA), non-adjuvant (NA) vaccines and physiological saline as controls, repeated at 28-day-old. On days 7,14,21 and 28 after the first vaccination, blood was sampled for determination of lymphocyte proliferation and serum antibody titer. In ND immunologic test, ND vaccines were made into high, medium and low dose EPI adjuvant, oil adjuvant (OA) and non-adjuvant (NA) vaccines contained equivalent ND antigens with inactivated antigens, OA materials and water for injection. Three hundred 14-day-old chickens were grouped, and the treatment and determinations were the same as the AI immunologic test. The results showed that EPI at high and medium doses could significantly promote lymphocyte proliferation and enhance serum antibody titer, its effect was better than that of non-adjuvant and oil adjuvant. These results indicated that EPI could enhance the immune effect of AI vaccine and ND vaccine, and could be used as the adjuvant of vaccines.Experiment Ⅷ. Determination of anti-immunosuppressive action of EPI The immunologic function of EPI on chickens with immunosuppression induced by cyclophosphamide was determined. Three hundred 11-day-old chickens were randomly divided into 6 equal groups and intramuscularly injected with 0.5 ml of cyclophosphamide (8 mg/ml) except for the blank control group, once a day for three successive days. At 14 days old, three EPI groups were intramuscularly injected with 0.7,0.5 and 0.3 mL of EPI respectively; drug control group were injected with 0.3 mL of astragalus polysaccharide injection; model control group and blank control group were injected with the same dose of physiological saline, all for three successive days. On days 7,14,21 and 28 after adminstration,6 chickens were sampled randomly from each group for determination of lymphocyte proliferation by MTT method, the body weight and indexes of immune organs were also measured. The results showed that the A570 value, average weight and the indexes of immune organs in high and medium doses of EPI groups were significantly higher than model control group on each time point after the first vaccination, and higher than APS group at most time points. It indicated that EPI had stronger antagonism on immunosuppression, and could use to prevent and cure the diseases of immunosuppression.Experiment Ⅸ. Study on action mechanism of EPI Two dosage forms of EPI on expression of IL-2 and IL-6 mRNA and secretion of Ig G and Ig M were determined. In cytokine expression test, two dosage forms of EPI with three concentrations (15.625,7.813 and 3.907μg·mL-1) were added into the cultivating system of chicken peripheral, taking PHA and cell as control. After cultivation of 12 h, lymphocyte were collected, total RNA was extracted, and then reverse transcription. The effect of them on expression of IL-2 and IL-6 mRNA was determined by fluorescence quantitative PCR. The results showed that two dosage forms of EPI could significantly promote the expression of IL-2 and IL-6 mRNA, the effect of suspension was better than watery solution. In Ig secretion test, two dosage forms of EPI with three concentrations were added into the cultivating system of chicken splenic lymphocyte singly or synergistically with LPS, taking LPS and cell as control. After cultivation of 24 h, supernate was collected, and the contents of Ig G and Ig M were assayed by ELISA. The results indicated that the two dosage forms of EPI could significantly promote the secretion of Ig G and Ig M. The effect of suspension was better than watery solution. |
PDF Full Text Request