| The BE can be used for the treatment of Duck Virus Hepatitis(DVH),which is composed of the three Chinese traditional herbs of Scutellaria baicalensis,Wild chrysanthemum flower and Epimedium.We decocted them in oral agent as the proportion of the three herbs,and the flavonoid compounds were its effective constituent.According to the regulations of veterinary medicine issued by Ministry of Agriculture,all the veterinary medicines should have a certain clinical effect,a safe and containable quality was also very important,and their microbial limit should be pass the test.So,we did some experiments as follows:1.The efficacy comparison of three different formulas of BE oral liquid for DVHIn order to compare the effects of flavonoid extracts and water decoction of the crude drugs,we designed the clinical test.Except the ducks of blank control group were isolated feeding(30 ducks),all the other 298 ducks of 6-days-old were given 0.2 mL DHAV diluent by intramuscular injection.Then we divided the 298 ducks into five groups at random:virus control group(58 ducks),chemical drug group(60 ducks),compound group-1(flavone compound group,60 ducks),compound group-2 and compound group-3(60 ducks respectively).Each duck was given 1.25 mg ribavirin every day in the chemical group,the ducks of compound group-1 were given 3 mg flavone of BE for each one,per duck in the compound group 2 and 3 was given 0.2 mL compound-2 and compound-3 respectively,and each of the duck in blank control was given the same cosolvent with BE,the treatment was carried for five days.After given the DHAV diluent 4 h,8 h and 54 h,we chose 5 ducks in every group and obtained their anticoagulant plasma and hepatic tissue as samples.We observed and recorded the survival rate of each group.We chose the best effect group to measure their glutathione peroxidase(GSH-Px),nitric oxide synthase(NOS),superoxide dismutase(SOD),catalase(CAT),and malondialdehyde(MDA)of plasma and liver to estimate the mechanism of the drugs' effect.The results showed that ribavirin,compound-1(flavone compound),compound-2 and compound-3 all can increase the survival rate and reduce the oxidative damage caused by DVH.The best curative effect was compound-1,and followed by compound-3.The above-mentioned indexes of compound-1 and chemical drug group were closed to the blank control group.The changes of each index in the liver appeared earlier than the indexes in the plasma.The results showed that the three formulas of BE all have a well effect for DVH,which may have something to do with the mechanism of anti-oxidative damage.2.Established the method for the content determination and identification of the effective constituent of Chinese herbal medicinal oral agents of BEOur purpose was that ensuring the BE oral liquid has a quality,safety and efficacy on clinic,we studied the quality of the BE,which included the content determination and identification the main ingredients in BE:using the phenol-sulfuric acid method to measure the saccharide content,the rutin method to detect the total flavones in BE,high-performance liquid chromatography was used to assay the content of baicalin and icariin in the BE,and we also used the thin-layer chromatography(TLC)to identify the baicalin,epimedium,wild chrysanthemum flower of BE.The studied showed that saccharide content was between 14.966 mg/mL and 15.215 mg/mL in the BE suspension,the total flavones of BE was at the range of 14.966?15.215 mg/mL,the contents of baicalin and icariin were at the range of46.566-47.507 mg/mL and 1.055?1.104 mg/mL respective.The rutin method,phenol-sulfuric acid method and HPLC methods of baicalin and icariin were proved to be practical and systemic.In our study,the TLC method of baicalin,epimedium,wild chrysanthemum flower we established not only were simple,convenient,but also had a good repetitiveness and a wide range of application,the three ingredients of BE were identified well.3.Research on the stability of Chinese herbal medicinal oral liguid of BEIn the experiment,we used the character,redispersibility,relative density,pH value,the total flavonoids content,the content of baicalin and icariin as the indexes to estimate the stability of effective constituent in the BE.And we also used the TLC to identify the baicalin,epimedium,wild chrysanthemum flower in every batch of BE.The stability of drugs includes the study of influence factors(such as the light and moisture so on)and the long-term experiment.The light acceleration test was served under the circumstance that the illumination intensity was 45001x ± 5001x,and we observed the above indexes of BE at 0 d,6 d,10 d and 14 d.Temperature and humid accelerated experiment with the conditions at 37 ?±2? and the humid of 60%±5%was used to observe 4 batches of BE suspension respectively at 1,2,3 and 6 month.And the long-term experiment was conducted in the condition of normal temperature and moisture at the 1,2,3,6,9,12,18 and 24 month to measure the above indexes of every batch of BE.As a result,we can see that the contents of baicalin and the total flavone were reduced slightly and the contents of icariin was almost stabilized in the light acceleration test and temperature and humid accelerated stability experiment,which suggest that the BE should be preserved under normal humid environment and avoided the direct sunlight.In the long-term experiment,the above indexes of every batch BE were stability.In the three experiments,the character,redispersibility,relative density and pH value of all the batches samples were stabilized.4.The establishment of the methods and testing for the BE' microbial limit testIn order to improve the quality of veterinary medicine and ensure its safety,we did the microbial limit test as the request of Chinese Veterinary Pharmacopoeia(2010 Edition).We chose the bacillus subtilis,staphylococcus aureus,escherichia coli,candida albicans and aspergillus niger as the positive control bacterium.According to the rules of recovery of bacterium we compared the membrane filtration method and the culture medium diluted method in the microbial limit test for BE oral liquid.As a result,there was no microbial in the BE suspension.The results we observed was impacted because of the preparation and concentration of the BE suspension when we chose the membrane filtration method.And the culture medium diluted method was appropriate for the detection of all the positive control bacterium but escherichia coli,and the recovery of staphylococcus aureus,bacillus subtilis,candida albicans and aspergillus niger were conformed to the Chinese Veterinary Pharmacopoeia(2010 Edition).The medicine have some antimicrobial effect for the escherichia coli,but there was also no bacterium include escherichia coli in BE oral liquid in this case. |
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