| The animal viral infectious diseases have been seriously endangering the development of animal husbandry in China,Up to now,there are not effective drugs to treat these diseases,therefore vaccination remains the most effective biomedical approach for the prevention of these diseases.However,most vaccines generally require the addition of an adjuvant to be effective.Due to the disadvantages of the common immunopotentiators,it becomes so urgent to study and develop a new-type immunopotentiators with high efficacy and low toxicity.Many researches indicated that many traditional Chinese medicine polysaccharides and their sulfated modification products possessed obvious immune-enhancing action.So,polysaccharides and their sulfations are regarded to produce as immunopotentiators.On the basis of successful research of compound astragalus polysaccharide?APS?and sulfated epimedium polysaccharide?sEPS?injection?AEI?,its peroral dosage form was prepared and named AEO in this study.Then,a series of studies were carried out including the production process,quality control,stability,safety,dosage,the effect and mechanism of systemic and mucosal immune enhancement.The details are divided into seven parts as follows:Experiment ?.Study on the production process of AEO On the basis of the technology of AEI,the preparation conditions of sulfated Epimedium polysaccharides?sEPS?,the content of crude drug and the dosage of preservative were studied.In preparation test of sEPS,the effects of precipitating and non-precipitating on the yield and carbohydrate content of sEPS were compared.The results showed that the yield and carbohydrate content of sEPS by non-precipitated were significantly higher than those by precipitated.So the process of precipitating were left out of the preparation of sEPS.In screening test of the content of crude drug,we compared the differences of three AEO preparations which with the crude drug content of 0.5,1.0,1.5 g·mL-1 after standing at room temperature for 1 month,taking the characteristics and stability of AEO as the index.The results showed that the AEO with the crude drug content of 0.5,1.0 g·mL-1 were transparent and had a small amount of precipitate which would be redissolved by shaking gently.So 1.0 g·mL-1 was selected as the crude drug content of AEO.In screening test of preservative,the differences of 4 AEO preparations which have been added different preservatives were compared after standing at room temperature for 3 months,taking the characteristics,stability,pH value and microbial limit as the index.The results showed that the AEO added 0.3%sodium benzoate was transparent and had a small amount of precipitate which would be redissolved by shaking gently.Moreover,the result of microbial limit test was qualified.So the option of preservative in AEO was 0.3%sodium benzoate.All the results of above three experiments considered,the production process of AEO was determined finally.Experiment ?.Study on quality control of AEO The quality control methods of AEO were studied.Taking thin layer chromatography?TLC?method to identify astragaloside,the chromatogram effects of three kinds of sampling volume were compared.The results showed that the clarity of spot was increased along with the increasing of sampling volume.Finally,5 mL was chosen as the optimal sampling volume.In polysaccharide assaying test,the phenol-sulphuric acid method was established and validated.The results showed that the maximum absorption peak of sample was at 490 nm wavelength,and there were good linear relationship at 0.01?0.05 mg·mL-1 of reference substance,high accuracy,high recovery rate,good reproducibility,and the sample solutions kept stable in 2 hours,which indicated the method was feasible to assay polysaccharide contents in AEO.In sulphate assaying test,the ultrasound-acidic barium chromate method was established and validated.The results showed that the maximum absorption peak of sample was at 375 nm wavelength,and there were good linear relationship at 0.01?0.03 mg·mL-1 of reference substance,high accuracy,high recovery rate,good reproducibility,and the sample solutions kept stable in 2 hours,which indicated the method was feasible to assay the sulphate contents in AEO.In astragaloside assaying test,the HPLC-ELSD method was established and validated.The results showed that there were good linear relationship at 0.04?0.4 mg·mL-1 of reference substance,high accuracy,high recovery rate,good reproducibility,and the sample solutions kept stable in 10 hours,which indicated the method was feasible to assay the astragaloside contents in AEO.All the results indicated that the above methods were scientific and reasonable,they could be used to control the quality of AEO.Experiment?.Study on stability of AEO In accordance with the guiding principles of traditional chinese veterinary medicine stability test technology,the stability of AEO was determined.In long-term test,the changes of indexes of three batches of AEO were detected in 0-12 months,including exterior colour,clarity,pH value,the content of polysaccharides,sulfate and astragaloside.The results showed that the content of polysaccharides were in 175.99?144.62 mg·mL-1,the content of sulfate were in 9.03?7.05 mg·mL-1,the content of astragaloside were in 53.56?41.39 ?g·mL-1,the pH value were in 6.23?5.82,the exterior colour and clarity also met the criteria.In accelerated test,the changes of indexes of three batches of AEO were detected in 0?6 months,including exterior colour,clarity,pH value,the content of polysaccharides,sulfate and astragaloside.The results showed that the content of polysaccharides were in 175.99?145.36 mg·mL-1,the content of sulfate were in 9.03?7.14 mg·mL-1,the content of astragaloside were in 53.56?42.25 ?g·mL-1,the pH value were in 6.23?5.76,the exterior colour and clarity also met the criteria.In light-accelerated test,the changes of indexes of AEO were detected after standing at 4500lx±1500 lx light intensity for 10 days,including exterior colour,clarity,pH value,the content of polysaccharides,sulfate and astragaloside.The results showed that there were no obvious changes in all indexes.All the results indicated that the stability of AEO was good and AEO was not sensitive to illumination.Experiment?.Determination of safety of AEO In accordance with the guiding principles of veterinary medicine test technology,the acute toxicity,long-term toxicity and target animal safety of AEO was determined.In acute toxicity test,Based on that the LD50 could not be detected,the maximum dose of AEO was detected.The results showed that the maximum dosage was 18000 mg·kg-1?polysaccharides?,or 120 g·kg-1?crude drug?.In long-term toxicity test,80 mice were randomly divided into 4 groups.The mice in three experimental groups were oral administrated with 0.4 mL AEO respectively at three doses,in blank control?BC?group were oral administrated with 0.4 mL normal saline,once a day for 14 successive days.On days 1,14 after the last administration,the body weight,hematological indexes,blood biochemical indexes,the heart,liver,spleen,lung,kidney index and the liver,kidney histopathological examination were measured.The results showed that all the indexes in three experimental groups had no significant differences with BC group.In target animal safety test,80 healthy chickens were randomly divided into 4 groups.The chickens in three experimental groups were taken orally with 1 mL AEO respectively at three doses,once a day for 9 successive days;the blank control?BC?group were not administrated.On days 1 before administration and 1,14 after the last administration,the body weight were measured;On day 1 after the last administration,hematological indexes and blood biochemical indexes were measured.The results showed that all indexes in three experimental groups had no significant differences with BC group except the RBC and HB in AEO1 were significantly higher than that of BC group.All the results indicated that AEO was non-toxic and safe for chickens.Experiment ?.The screening test of AEO dosage The immune enhancing effect of 7 doses of AEO were compared.300 14-day-old chickens were randomly assigned into 10 groups and vaccinated with ND vaccine except for blank control?BC?group,repeated vaccination at 28 days old.At the same time of each vaccination,the chickens in 7 experimental groups were taken orally with 1 mL AEO at 2,4,6,8,10,12,14 mg·mL-1 respectively,once a day for three successive days;in injection control group were injected with AEI once,and in vaccination control?VC?and BC groups were not administrated.The spirit,activity,ingestation,drinking,pathogenic and dead statuses of chickens were observed every day after administration.On days 7,14,21 and 28 after the first vaccination,the body weight and serum ND-HI antibody titer were measured.The result showed that after administration,the chickens in each group had no any untoward effect,the antibody titers and average weight in 4,6,8,10 mg groups were higher or significantly higher than VC group,and there were no significant differences between these four groups and AEI group.The effect of 6 mg group was the best,the next was 4 mg.Considering the clinical application,5 mg was determined as the best dosage of AEO.Experiment?.Study on the systemic immune-enhancing action and mechanism of AEO The adjuvanticity of AEO was determined in test 1.320 14-day-old chickens were randomly assigned into eight groups and vaccinated with ND vaccine except for blank control?BC?group,repeated vaccination at 28 days old.At the same time of each vaccination,the chickens in three experimental groups were taken orally with AEO respectively at three doses,in two component control groups with APS and sEPS,once a day for three successive days;in injection control group were injected with AEI once,and in vaccination control?VC?and BC groups were not administrated.On days 7,14,21,28 and 35 after the first vaccination,peripheral lymphocyte proliferation,the serum antibody titer,IFN-? and IL-2 concentrations and on day 35 immune organ index were measured.The results showed that AEO could significantly promote lymphocyte proliferation and development of immune organ,enhance antibody titer and IFN-? and IL-2 concentration,the effects of high and medium dosage groups were slightly stronger than that of AEI,but significantly stronger than those of two components groups.The anti-immunosuppressive action of AEO was determined in test 2.180 11-day-old chickens were randomly assigned into 6 groups and intramuscularly injected with 0.5 mL of cyclophosphamide(8 mg·mL-1)except for the blank control?BC?group,once a day for 3 successive days.At 14 days old,three AEO groups were taken orally with AEO respectively at three doses,AEI control group were injected with 0.5 mL of AEI,once a day for 3 successive days;Model control group and BC group were not administrated.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 AEO groups were significantly higher than those in model control group on 14?28 d after the first vaccination.All the results indicated that AEO could enhance the immune effect of ND vaccine and antagonize the immune suppression induced by cyclophosphamide.Experiment ?.Study on the mucosal immune-enhancing action and mechanism of AEO 280 14-day-old chickens were randomly assigned into 7 groups and vaccinated with ND vaccine except for blank control?BC?group,repeated vaccination at 28 days old.At the same time of each vaccination,the chickens in three experimental groups were taken orally with AEO respectively at three doses,in two component control groups with APS and sEPS,once a day for three successive days;in vaccination control?VC?and BC groups were not administrated.On days 7,21 and 35 after the first vaccination,six chickens were selected randomly from each group for measurements of the sIgA contents of the trachea,slgA and IL-17 contents of the washing liquors of the duodenum and jejunum,counts of the lymphocytes in the duodenal endothelium and counts of the IgA+ cells in the jejunal endothelium and cecum tonsil.The results showed that AEO significantly promoted the secretion of slgA and IL-17,increased the numbers of lymphocyte and IgA+ cells.All the results indicated that AEO could enhance the respiratory tract and intestinal mucosal immunity. |
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