| Immunity level is a significant indicator of animal health,it can not only play a crucial role in the prevention and control of animal epidemic diseases but also have great impacts on animal productivity.With the development of animal husbandry,the environments for animals to grow have been changed result from scaled raising.Diseases of livestock and poultry are going to be increasingly complex and difficult to defend because of high stocking density,poor air quality and other factors.The application of abundant vaccines and medicines hence becomes a key part of scaled raising to control diseases.However,the harms which caused by the abuse of antibiotics and chemosynthesis drugs are gradually valued by people and the ways to cut the use of these drugs inevitably become the problem that needs to be solved by breeding industry.Therefore,the way to decrease morbidity and the application of antibiotics via improving immune system and disease resistance in animal bodies has been an important filed in current studies of animal husbandry.Numerous studies have demonstrated that many herbs possess immunoregulatory properties which could activate immune system,pep up immune cells and increase antibody production.For example,Astragali Radix and Codonopsis Radix,common Chinese traditional medicines used to benefit vital energy,could improve the immunity of animal bodies.But given the price of Codonopsis Radix,it costs too much to apply to animal husbandry,therefore it is awfully hard to be adopted or popular in livestock and poultry production.According to some research findings,immunological substances like polysaccharide and lobetyolin,which contained in the stems and leaves of Codonopsis Radix,act like the Radix.They can almost work as well as the Codonopsis Radix,provided that the active ingredients are extracted in an appropriate way.Some studies in animal husbandry also reported that stems and leaves of Codonopsis Radix used as feed additives improved the production performances.In this study,we chose the basal part of stems and the leaves of Codonopsis Radix together with Astragali Radix as raw materials to prepare the immunopotentiator the Codonopsis Pilosula stems and leaves and Astragalus powder.We carried out a serious of systematic experiments to research the production technology of the Codonopsis Pilosula stems and leaves and Astragalus powder.Moreover,quality control,drug safety,pharmacodynamics and adjuvanticity to the vaccines designed for swine or poultry diseases were also taken into consideration.The aim of this study was to manufacture the compound preparation with security,effectivity and affordability which could function as traditional Chinese veterinary immunopotentiator,furthermore,it could provide evidences that the Codonopsis Pilosula stems and leaves and Astragalus powder would help vaccines to prevent and control the animal diseases.The experiments were mainly divided into six parts.Experiment Ⅰ Study on production technology of the Codonopsis Pilosula stems and leaves and Astragalus powderIn order to optimize the production technology of the Codonopsis Pilosula stems and leaves and Astragalus powder,the extraction yield of polysaccharides from Astragali Radix and Codonopsis Radixwere chosen as the investigate indicators together with AstragalosideⅣ from Astragali Radix.The optimal extraction process of the Codonopsis Pilosula stems and leaves and Astragalus powder was designed by orthogonal test.In this experiment,the spray drying process conditions were also selected.The results showed that the best extraction process of the Codonopsis Pilosula stems and leaves and Astragalus powder was treating the materials with water which is 8 times over materials and soaking for 30 minutes,then boiled and reflux extracted for 30 minutes followed by filtration.The residue was retreated twice as described above.The resulting filtrates were combined and concentrated under reduced pressure at 60℃ to a relative density of 1.01(60℃).The inlet air temperature and the liquid volume during spray drying process were set as 150℃and 120 mL/min,respectively.The Codonopsis Pilosula stems and leaves and Astragalus powder was then prepared.Experiment Ⅱ Study on quality control and stability of the Codonopsis Pilosula stems and leaves and Astragalus powderIn order to establish the quality control method of the Codonopsis Pilosula stems and leaves and Astragalus powder and to investigate their stability,the identification method and content determination method of active ingredients in the extracts were studied in the experiment,the stability of the three batches of samples was also investigated.Firstly,Astragaloside Ⅳ and Lobetyolin,the main components of the compound,were identified by thin-layer chromatography(TLC).The content of the main components,AstragalosideⅣ and polysaccharide,was determined by high performance liquid chromatography(HPLC).Secondly,the stability of the material extracts was determined by isothermal accelerated test,light acceleration test and retention observation test.The results showed that under the established TLC conditions,the fluorescent spots at the corresponding positions of the chromatogram could be detected to display the same color of the sample and the reference substance,besides,the established content determination method for Astragaloside Ⅳ and polysaccharide has high precision,reproducibility and stability.The content of Astragaloside Ⅳ in the three batches of samples was 0.20%,0.19%,and 0.20%,respectively;the content of Astragaloside polysaccharide was 75.75%,75.34%,and 74.68%,respectively.The content limit is tentatively set as follows:the content of Astragaloside Ⅳin this product shall not be less than 0.18%,and the content of polysaccharide(based on anhydrous glucose,C6H12O6)shall not be less than 70%.The stability test results showed that in the accelerated stability test within 6 months,the long-term stable experiment within 12 months,and the light acceleration test during 10 days,the three batches of samples showed hardening agglomeration in addition to the accelerated stability test.There was no significant change in the identification and content of other major components.In addition to the phenomenon of hardening agglomeration in the accelerated stability test,three batches of samples did not show clear changes in the other experiments,which indicated that the material extracts were basically stable during the initial stability test.However,the phenomenon of hardening agglomeration in the accelerated stability test suggested that the samples should be stored in a cool dry place after they were sealed.Experiment Ⅲ Study on acute toxicity and subchronic toxicityof the Codonopsis Pilosula stems and leaves and Astragalus powderIn order to study the safety of the Codonopsis Pilosula stems and leaves and Astragalus powder,acute toxicity and subchronic toxicity of the extracts were measured.First of all,in multiple pre-tests,neither the absolute lethal dose(LD100)nor the median lethal dose(LD50)on mice caused by the material extracts,were able to be determined,the maximum tolerance dosage was hence determined.Secondly,according to the clinical dosage and the maximum tolerance dosage,the compounds of the material extracts were divided into three dosage levels:high,medium and low.The mice were then administered with each dose of material extracts by gavage in 28 days to measure the subchronic toxicity.The results showed that the maximum tolerance dose of the extracts in mice was 48 g/kg·W.The results of the subchronic toxicity test showed that the discomfort performances were observed onlyafter the first three times of drug administration.The percentage of lymphocytes in the experimental group was significantly higher than which in the control group on the first day after drug withdrawal,and the other hematological indicators did not show significant differences.No death occurred in each group of mice,and there were no significant differences in average weight gain,organ index,and serum biochemical parameters.No significant pathological changes were observed by necropsy and histological observation.The results showed that the Codonopsis Pilosula stems and leaves and Astragalus powder had no acute toxicity and significant subchronic toxicity,and it is safe for clinical application.Experiment Ⅳ Pharmacodynamics of the Codonopsis Pilosula stems and leaves and Astragalus powder.In order to study the immune enhancement of the Codonopsis Pilosula stems and leaves and Astragalus powder,the effects of them were studied on improving cellular and humoral immunity of chickens in vitro and in vivo.In vitro experiment,firstly,the safe concentration of the Codonopsis Pilosula stems and leaves and Astragalus powder on chicken peripheral blood lymphocyte was confirmed,and then the effect of different concentration on peripheral blood T-lymphocyte proliferation was determined.In vivo test,the material powder was administered to chickens in 7 doses of drinking water,and the control group and blank control group were set up.Then effects of the material powder on the immune enhancement of Newcastle disease vaccine and the weight of chickens were observed.The results showed that the maximum safe concentration of the material powder on Chicken Lymphocyte in vitro was 7.813 μg·ML-1.The maximum safe concentration of the material powder alone could significantly stimulate lymphocyte proliferation in the range of 7.813-0.016 μg·mL-1,and the maximum safe concentration of the Codonopsis Pilosula stems and leaves and Astragalus powder has a strong effect on enhancing cellular immunity.In vivo test showed that the powder could significantly increase serum antibody titer and enhance humoral immunity.What’s more,they could increase daily average weight gain,and had no adverse effect on the growth and health of chickens.Experiment Ⅴ The immunoenhancement effect study of Codonopsis Pilosula stems and leaves and Astragalus powder on Newcastle disease vaccine in chickensIn order to observe the immune enhancement effect of the Codonopsis Pilosula stems and leaves and Astragalus powder on Newcastle vaccine,540 non-immune Lohmann hens of 2 weeks old were random divided into six groups,90 in each group.The control group did not take any treatment and was raised.The other 5 groups were all immunized with the Newcastle disease IV lineage vaccine by eye drops and nasal drops.Three experimental groups began to drink the solution of Codonopsis Pilosula stems and leaves and Astragalus powder with 3 dosage levels,the drug control group was administrated with astragalus polysaccharide powder for 7 days at the same time,and the immune control group was not administered.The chickens were booster immunized and administrated at 28 days.Blood samples were collected at 1,2,3,and 4 weeks after the first immunization and serum NDVHI antibody titers and peripheral blood lymphocyte proliferations were measured.The results showed that the high and medium doses of the Codonopsis Pilosula stems and leaves and Astragalus powder could significantly increase the proliferation of peripheral blood lymphocytes and increase the HI titer in serum,meanwhile showed a certain growth-promoting effect,which was superior to the control drug.Since there is no significant difference between high dose and medium,medium dose was recommended as clinical application.Experiment Ⅵ Clinical research of the effect of the Codonopsis Pilosula stems and leaves and Astragalus powder on enhancing the immune effect of Swine fever vaccineIn order to observe the immune enhancement effect of the Codonopsis Pilosula stems and leaves and Astragalus powder on the swine fever vaccine,150 20-day-old piglets were randomly divided into 5 Groups,30 piglets in each group and each group was immunized with swine fever vaccine.At the same time,the three drug dose groups were feeding with the Codonopsis Pilosula stems and leaves and Astragalus powder at a ratio of 0.3%,0.2%and 0.1%,and the drug control group was feeding with Astragali Radix polysaccharide powder at a ratio of 0.1%for 3 consecutive days;Vaccine control group is not administered;the second immunization was performed 30 days after the first immunization,and the above administration was repeated for 3 days.At 10(D10),20(D20),30(D30),40(D40),50(D50),60(D60),70(D70)days after the first immunization,six piglets was randomly selected in each group.Blood samples were collected from the ear vein and serum was separated.The antibody titers of the piglet were determined by positive indirect agglutination according to the kit instructions.Meanwhile,six piglets in each group were randomly selected in each group for weighings,and the monthly average weight gain of each group was counted.The results showed that high and medium doses of the Codonopsis Pilosula stems and leaves and Astragalus powder could significantly increase serum antibody titer and daily weight gain,which were better than Astragali Radix polysaccharide powder.The antibody titer of the high dose group and the daily weight gain of the middle dose group in the early stage were the highest.In the late middle dose group,the antibody titer of the middle dose group and the highest daily gain of the high dose group in the later stage were the highest.And there was no significant difference between the high and middle doses.The medium dose was recommended as the clinical application dose.In conclusion,we researched the Codonopsis Pilosula stems and leaves and Astragalus powder for its production technology,quality control,drug safety,pharmacodynamics and adjuvanticity to the vaccines designed for NDV and CSFV.According to the research results,we established a serious of appropriate production technology and quality control standards.The Codonopsis Pilosula stems and leaves and Astragalus powder has been proved to be safe and non-toxic and it also has a certain promoting effect both on humoral immunity and cellular immunity.Additionally,the Codonopsis Pilosula stems and leaves and Astragalus powder would significantly improve the protective effect of the vaccines designed for chicken and swine diseases in clinical applications.The growth promoting effect can be developed as an animal immune enhancer with the form of traditional Chinese veterinary medicine or feed additive. |
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