| The compound prescription of HG is composed of Hypericum japonicum Thumb and Glycyrrhiza uralensis Fisch.We have confirmed that the compound has good therapeutic effect to the duck viral hepatitis at preliminary studies of the experiment.For further development and utilization of the compound mixture,in this research,we did some experiments to evaluate the safety,quality and the effect on relieving fever of the Chinese herbal medicinal oral solution based on the declaration-principles of the new veterinary drug such as the drug safety and reliable,quality control,stable quality,etc.The main contents are as follows:Experiment Ⅰ Study on production technology of Hypericum japonicum Thumb and HGIn this study,orthogonal experiments(four factors and three levels)were used to optimize the extraction of flavonoid from Hypericum japonicum Thumb by ethanol as solvent(hot ethanol refluxing extraction),at 510 nm,the absorbent was measured to calculate the quality of flavonoid by ultraviolet spectrophotometry.In order to investigate the intersection of the four main factors such as the extraction temperature(70℃,80℃,90℃),the extraction time(2h,3h,4h),ethanol concentration(30%,40%,50%)and the material liquid ratio(1:30,1:40,1:50),therefore,the orthogonal experiment was designed to determine the best extracting technology of Hypericum japonicum Thumb.The results of our studies indicated that the maximum of flavonoid was 108.69 mg/g when the extraction temperature was 80℃,extraction time was 3h,50%ethanol and the ratio of material to liquid was 1:40.When the best extraction technology of Hypericum japonicum Thumb was determined,we mixed up the residue with the rest of Chinese herbs of the compound prescription,and then,we could extract all kinds of flavonoid by boiling of water-soluble herbal substances as the same way like former.The several main factors and levels of this orthogonal experiment contain solid-to-liquid ratio(1:10,1:15,1:20),decoction time(40min,60min,80min)and decocting frequencies(1,2,3).As we can see from the experimental data that we got an optimal manufacturing technique of the compound Chinese herbal prescription.The maximum of flavonoid is 42.5 mg/g through a series of certification testing when the decoction time is 40min and the decoction frequency is twice,solid-to-liquid ratio is 1:20,meantime,we verified the extraction technology of HG is practical and feasible.Experiment Ⅱ Evaluation on the safety of compound Chinese herbal oral solution of HGIn the preliminary experiment of acute toxicity test,30 ICR mice,weighing 20g,were divided into three groups:high dosage group,low dosage group and blank control group,all groups were administered by intragastric administration(0.8ml/20g).The concentration gradient of the two groups was 2g/ml and 0.2g/ml respectively.The control group was administered by saline as the same way.The status including behavior,drinking,feeding and feces was observed and recorded for 7 days after intragastric administration.The results showed no death of mouse was found,so we failed to detect the LD50 of HG.For this reason,we investigated the experiment of maximum administration dosage.Total 20 mice weighting 20g,were divided into the compound administration group and blank group,and were separated breeding,adaptive-breeded about a week before the formal experiment.And the same time fasting the water overnight is necessary,the administration group was administrated 2g/ml of HG according to the maximum tolerance of mice with the dosage of 0.8ml/20g body weight,the blank control group was administered by saline as the same way.The treatments were repeated 2 times one day,with an interval of 8h.Based on the detail observation and records such as the drinking water,breathing,feeding,exercise and death,we continuously monitored for 7 days.The results showed that all mice in the group were normal,maximum administration dosage of HG is 160 g/kg(calculated with net content),and it is 56 times as large as the clinical treatment of duck daily.During the long-term toxicity test,80 Wistar rats weighting about 200g as the object for investigation,were randomly divided into 4 groups.Drug groups were given traditional Chinese medicine HG with the volume of 2.4ml at the concentration of 2g/ml,0.67g/ml,0.22g/ml respectively,the control group was given the same volume of saline,once a day,continuous gavage for 30 days.And we continuously observed 14 days after stopping gavage.The body weight of each group was weighed before administration(D0),the first day after drug withdrawal(D1),7d(D7)and 14d(D14).During the period of test,the Wistar rats’ spirit,activity,ingestion,drinking,morbidity and mortality were sequentially observed.For the sake of measuring the blood routine,biochemical criterions of hepatic,renal and many other internal organ function,we selected randomly 10 Wistar rats at D7 and D14,male and female half,blood was drawn from femoral artery.At the same time the weight of heart,livers,spleen,lungs,kidneys,testes/ovary,stomach and intestines were recorded in detail and the pathology changes were observed.The results displayed that the clinic indication,mental state of the mice were normal.Blood routine data and blood biochemical indexes of each group were within the scope of normal values after using by HG.The pathological changes of visceral organ were not found by naked eye.The sections of livers,kidneys and many other organs showed no significant change compared with that of the blank control under the view of microscope.All of results indicated that the compound prescription of HG had no obviously long-term toxicity.Experiment Ⅲ Preliminary research on the antipyretic effect of HG on rats fever model induced by dry yeastWistar rats weighting about 200g were selected as the experimental animals.The data were measured and recorded according to the basal body temperature.The rats were divided into blank group,model group and high,medium and low administration group.The dosage of 10mL/kg dry yeast suspension at the concentration of 20%,was administrated to the model group and the rats in the administration groups.After 2 hours,the rats of the high,medium and low dosage groups were treated with 2.4ml HG at the concentration of 2g/ml,0.67g/ml and 0.22g/ml,respectively.The rats in the model group and the blank group were treated with saline at the same volume of 2.4ml/200g.After 6 hours of administration,4 rats were sacrificed in each group,and the rats were anesthetized with ether.Blood of the femoral artery was used to detect the hematology and serum biochemical indexes.The same treatment was repeated one time at the next day.During the experiment,the rectal temperature was measured once every 2 hours,and the clinical signs such as mental state,behavior,drinking,feeding,breathing,death and autopsy were recorded.The results showed that the body temperature of rats treated by dry yeast began to drop at 2 hour,get together,drowsiness,and then expressed the performance of excitement,biting the back,hair chaos,breathing faster,tail hot,fecal dry knot and other abnormal pathological state.Autopsy changes of edema,bleeding spots were found in the liver and lungs.All the rats in the blank group showed no change.Analysis of blood and biochemical indicators found white blood cells and mononuclear phagocytic values of blank group were high,but the levels of aspartate aminotransferase and total bilirubin were low,the difference was significant,and the other indexes were within the normal range.There was no significant difference in liver and kidney organ indexes.The body temperature of the rats in the high and middle dose groups decreased gradually to the normal body temperature.At the same time,the rats in the model group were still high fever,indicating that the experiment was basically expected.Results showed that we got an initial clinical antipyretic effect by the Chinese herbal medicinal oral solution of HG. |
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