Studies On Chinese Herbal Medicinal Flavone Immunopotentiator EPO And Its Action Mechanism

In recent years,the prevalence of livestock and poultry epidemic deseases has had great impact not only on animal husbandry,but also on endanger public health security.These serious diseases mostly belong to viral infections,some of which belong to immune suppression of diseases.So far,there is no special valid medicine for treatment.In addition to strengthen management,application of vaccine immunization is the main preventive measures.Application of immunopotentiators to improve the immune effect of vaccine and strengthen the disease resistance of animals is one of the important measures to improve the effect of prevention.So the immunopotentiators have become a hot issue of research in recent years.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.Some research has shown that many of the traditional Chinese medicines?TCM?have good immune enhancement effect and have significant advantages.Based on the successful development of EPI to improve formulations and technology,this study was carried out including preparation technology,quality control,stability,safety,clinical application dosage,clinical effect and the action of immune enhancement and mechanism.The study is divided into eight parts as follows:Experiment ?.Study on the Preparation Technology of EPO Epimedium extraction and the solvent dosage were studied on the basis of preparation technology of EPI.In EE extraction test,epimedium polysaccharides were taken as an index to observe the influence of temperature and amount of water adding on polysaccharides content.The results showed that when 20-fold water was added and the temperature was 100?extraction rate was optimal and polysaccharides content was the highest.In the solvent dosage test,the impact of different dosage of anhydrous ethanol,granulesten,twain,polyethylene glycoland,glycerin on the properties and stability of the preparations was investigated.The results showed that the optimized proportion for the solvent was anhydrous ethanol 9.0%,twain 2.0%,glycol 8.0%,glycerin 6.0%,and granulesten 1.0%.Based on the above results,the EPO preparation technology is as follow:decoct epimedium with 20 times of water for 1.5 hour by twice for the first time,1 hour for the next;combine the filtrate,let it settle,centrifuge to remove sediment,add distilled water to 770ml,filtrate,add 60 ml glycerol to the filtrate,mix them together;add ethanol 90 ml,granulesten 10 g,Polyethylene glycol-400 80 ml to propolis,mix together,and slowly put them to the epimedium solution,mix again,after packaging and sterilization.Experiment ?.Study on Quality Control of EPO The quality standard study was studied In icariin identification test,three different sampling volume and three different sample spotting volume were compared,the results showed that when the sampling volume was 2.5 ml and sample spotting volume was 3 ?l,thin layer chromatography was the best.In norizalpinin identification test,three different sample spotting volume were compared,the result showed that when the volume was 3 ?l,thin layer chromatography was the best.In content assaying test of icariin,separation effect of icariin by 2 types of mobile phase and different elution method with HPLC were compared,and method validation was tested,the results stated that,using acetonitrile-0.15%phosphate?25:75?with the gradient elution,icariin content was analyzed quickly and accurately,and method validation proved that it possessed a good linear relationship,higher degree of precision and recovery,and good stability.In chrysin and norizalpinin content assaying test:the analysis method of chrysin and norizalpinin in EPO with HPLC was established.The results showed that chrysin and norizalpinin appear with good shape and without impurity interference or tail formation phenomenon.Method validation proved that chrysin and norizalpinin had a good linear relationship with the peak area in 150?750 ng;it possessed higher degree of precision and recovery,and good stability,which suggests this method was workable.Experiment ?.The Determination of the Stability of EPO The stability of EPO was determined.The changes in character,effective ingredient?icariin,galangin and chrysin?content,sedimentation volume ratio,pH value of EPO were observed with emphasis.In the factors test,in the light of 45001x±5001x irradiation,the changes of all indexes from three batches of EPO samples were detected on day 0,5 and 10,the results showed that the characters of three batches of EPO samples in days 10,the sedimentation volume ratio and PH value remained stable,and the redispersibility was favorable,the content of Icariin,chrysin and galangin slightly decreased,which indicated that EPO had certain sensitivity to light,and shoud be kept in dark place.In the accelerated stability test,the changes of all indicators of three batches of sample in 0,1,2,3 and 6 months on the condition of temperature 30?±2?,relative humidity 60%±5%,the results showed that three batches of samples remained stable in 6 months?In the long-term stability test,the changes of all indicators of three batches of samples in 0,3,6,9and 12 months on the condition of room temperature were observed,the results showed that three batches of samples remained stable in 12 monthsExperiment ?.Determination on Safety of EPO Safety of EPO was determined.In the test of acute toxicity,on the base that LD50 could not be determined in prediction experiment,the results of MLD of EPO was 32g/kg?equivalent to content of crude drug?.The results indicated that EFO was non-toxic in practice.In the test of long-term toxicity,100 ICR mice about 20g weight were randomly divided into five average groups,Three dose groups were given 40,10,2.5 mg/ml of EPO 0.4 ml respectively,solvent control group?SC?were given solvent which did not contain EPO 0.4 ml,the control group?BC?were given equal volume of saline,once a day for successive 14 days.Weighing the weight of mice before?D₀?,and 1?D₁?,7?D₇?and 14(D₁₄)days after administration,and calculating the average weight.On D₁ and D₁₄,the blood samples were collected to determine the hematology,serum biochemical indicators and viscera indexes.Cutting liver and kidney tissue,paraffin sections,HE staining,the pathological changes were observed by microscopy.The results showed that clinical signs of animals in each group were normal.On D₁ and D₁₄ after medicine stopping,the hematology and serum biochemical indicators of all treatment groups and SC group were normal,necropsy and liver and kidney biopsy had no significant pathological changes,which indicated that EFO had no obvious long-term toxicity.In the test of target animal safety in chickens,eighty 14-day-old healthy White Roman chickens were randomly assigned into 4 average groups.Three treatment groups were taken orally with 1.25 mg/ml,0.75 mg/ml and 0.25 mg/ml of EPO 0.4 ml respectively,once a day for nine successive days,and the control group?BC?were not administrated.The reaction of chickens was observed every day after the first administration until D7 of administration respectively.Weighing the weight of chickens on 0 day?D0?before administration,and D₁ and D7 after medicine stopping,and calculate the average weight.On D₁ after administration,the blood samples from four chickens randomly selected from each group were collected to determine the hematology and the content of serum ALT,ALP,creatinine?CREA?and UREA.The results showed that clinical signs of animals in each EPO group were normal,and there were no significant difference in all indexes with BC.These results indicated that EPO was absolutely safe to the target animal chickens.Experiment ?.The Screening Test of EPO Dosage Three hundred 14-day-old chickens were randomly assigned into 10 groups,30 for each group,except for the blank control?BC?group,vaccinated with NDV-IV vaccine by nose and eyes,revaccination at 28 days old.At the same time of each vaccination,chickens in seven groups dosed with EPO were taken intragastric administration respectively 1 ml with EPO of 0.25,0.5,0.75,1.0,1.25,1.5,and 1.75 mg/ml,once a day in three consecutive days.The chickens in EPI group were intramuscularly injected with EPI 0.5 ml once,in vaccination control?VC?group and BC group,no administration.On 7 day,14,21 and 28 after the first vaccination,six chickens chosen randomly from each group,were sampled blood from wing vein for determination of serum hemagglutination inhibition?HI?antibody titer of ND,weighing and calculating weight gain rates.The results indicated that,chickens of all groups kept normal in psychosis and diet.Antibody titer of 0.25 mg of EPO was the highest and significantly higher than VC group at all time points,and were significantly higher than other EPO groups at last two time points.The results revealed that EPO in suitable dosages could significantly enhance humoral immune response and promote the growth of chickens,and the dosage of 0.25 mg was the best,which was similar to the EPI.Experiment ?.The Determination of Enhancing Immune Effects of ND Vaccine with EPO 14-day-old chickens were randomly assigned into eight groups and,except for blank control?BC?group,vaccinated with ND vaccine,revaccination at 28 days old.At the same time of each vaccination,the chickens in high,middle,and low doses of EPO groups were taken orally respectively with EPO of 0.125 mg,0.25 mg and 0.5 mg,and the chickens in drug control group were taken orally respectively with EP and PF 0.25mg,once a day for three consecutive days.The chickens in EPI group were intramuscularly injected with EPI 0.5 ml once,in vaccination control?VC?group and blank control?BC?group,no administration.Serum antibody titer,lymphopoiesis,serum IL-2 and IFN-y and immune organ index were tested on 7,14,21,28 and 35 days after the first vaccination.The results showed that antibody titer at all time points,lymphocyte proliferation on D₁4?D35,and IL-2 and IFN-y content at most time points were higher or significantly higher than VC group,components of medicine control group and were a little better than EPI group,and the immune organs indexes in all time points improved a little.The results suggested that EPO could enhance the systemic immune response significantly,and effects of the middle and high dosage of EPO were better.Experiment ?.Determination of Enhancing the Intestinal Mucosal Immunity of Chickens 14-day-old chickens were randomly assigned into eight groups and,except for blank control?BC?group,vaccinated with ND vaccine,revaccination at 28 days old.At the same time of each vaccination,the chickens in high,middle and low doses of EPO groups were taken orally respectively with EPO of 0.125 mg,0.25 mg and 0.5 mg,and the chickens in components drug control groups were taken orally respectively with EP and PF of 0.25mg,once a day for three consecutive days.The chickens in EPI group were intramuscularly injected with EPI 0.5 ml once,in vaccination control?VC?group and BC group,no administration.On days 7,21 and 35 after the first vaccination,six chickens were selected randomly from each group to measure sIgA and IL-17 content in washing liquor of duodenum and jejunum,count the number of lymphocyte in duodenal endothelium and IgA+ cell in jejunal endothelium and cecum tonsil.The results showed in all time points of the first vaccination sIgA and IL-17 content in washing liquor of duodenum and jejunum,lymphocyte in duodenal endothelium and the number of IgA+ cell in jejunal endothelium and cecum tonsil were significantly higher than that of VC group and were higher or significant higher than EPI,EP and PF groups.This indicated that EFO could enhance the intestinal mucosal immunity.The effects of the high and middle doses of EPO were the best,and better than EPI slightly.Experiment ? Determination of Anti-immunosuppressive Action of EPO Three hundred healthy 11-old-day chicken were randomly divided into 6 groups in equal numbers;each chicken was intramuscularly injected 0.5 ml of 8 mg/ml cyclophosphamide,once a day for three successive days,except for the BC group;when the chickens were 14-day-old,the high,middle and low doses of EPO groups were taken orally with 0.5mg,0.25mg and 0.125 mg of EPO respectively,the EPI control group was injected 0.5 ml EPI once a day for three consecutive days,no administration to the MC and BC group.On 7,14,21,and 28 days after administration,4 chickens were randomly selected from each group to weigh,sampling blood from the heart,to test T lymphocyte proliferation of peripheral blood with MTT method.Meanwhile,6 chickens were randomly selected,weigh and calculate the average weight,cut part the spleen,thymus,bursa and neck to weigh,and calculate the index of immune organs.The results indicated that lymphocyte proliferation,immune organ indexes and the average weight in EPOH and EPOM groups and EPI group were higher than that of the MC group at most time points.The results indicated that EPO could significantly alleviate the immunosuppression induced by cyclophosphamide in chickens,and promote the growth of chickens.The effects of high and middle dose of EPO were the best and similar to EPI.