| Epimedium brevicornu Maxim is a perennial herb,also known as short-horned Epimedium,three-leaf clover,fairy spleen,horn flower,and so on.It is a plant of the genus Epimedium.Epimedium is an ancient Chinese medicine that has a certain improvement effect on impotence,premature ejaculation,backache,leg numbness,hemiplegia,neurasthenia,and tinnitus.The active substances of Epimedium are icariin,deoxymethyl icariin,8-dehydromethyl icariin,among which icariin has the most active ingredients.Cherax quadricarinatus,also known as "Australian freshwater lobster",with delicious meat,high nutritional value,large market demand and certain economic effects.The Cherax quadricarinatus shrimp is strong but has many diseases.In winter,the clam shell is easy to die.The aim of this study was to evaluate the acute toxicity of Epimedium extract in mice,the long-term toxicity to rats,the safety of Epimedium extract on Cherax quadricarinatus and experimental clinical trial on Cherax quadricarinatus,and provide a theoretical basis for the clinical application of Epimedium extract.1.Identification and content determination of Epimedium extractMethod: Identification of Epimedium extract was performed by thin layer chromatography and high-phase liquid chromatography,and determination of icariin carried out by ultraviolet spectrophotometry.Result: The sample was identified as thin extract of Epimedium by thin layer chromatography and high-phase liquid chromatography.The content of icariin in the Epimedium extract was 10% which determined by ultraviolet spectrophotometry.Conclusion: The sample is Epimedium extract,in which the content of icariin is 10%.2.Acute toxicity test of Epimedium extract in miceMethod: The acute toxicity test was carried out by intragastric administration for clinical observation and necropsy,and the extract of Epimedium extract was tested on mice.Median lethal dose and maximum tolerated dose.Result: The Epimedium extract without acute toxicity to mice,and its maximum dose is 20 g/kg ·BW.Conclusion: This Epimedium extract is non-toxic to mice.3.Long-term toxicity test of Epimedium extract on ratsMethod: 80 SD rats were randomly divided into 4 groups,including blank control group,high dose group(20 g/kg ·BW),medium dose group(10 g/kg ·BW)and low-dose group(5 g/kg ·BW),20 rats in each group,male and female,half-bred,10 cages per cage.The animals were treated for continuous medication for 30 days with daily clinical manifestations.On Day 30 after first treatment and Day 7 post last treatment,10 animals in each group were used for the determination of hematology,blood biochemical indicators and pathological examination of major organs respectively.Result: There were no abnormal changes in clinical manifestations,behavioral activities,feces,and so on.There were no toxic reactions and deaths in the rats in each group.The blood routine,blood biochemistry and other indicators of the animals in each group were observed.There was no significant difference between the index and the blank group(P > 0.05).There were no visual and histopathological changes in the heart,liver,kidney,spleen,gastrointestinal,testis and ovary of each group.Conclusion: This Epimedium extract is non-toxic in rats.4.Safety test of Epimedium extract against target animals(Cherax quadricarinatus)Method: 150 Cherax quadricarinatus with healthy and uniform weight were randomly divided into 5 groups,including 1 time dose group(addition with 0.1% Epimedium extract in feed),3 times dose group(addition with 0.3% Epimedium extract in feed),5 times dose group(feed supplement 0.5% Epimedium extract),10 times dose group(feed addition1.0% Epimedium extract)and blank control group.Each group consisted of3 parallels,10 tails each parallel,and was housed in separate boxes.After 7days treatments with Epimedium extract,symptoms observation,morphological parameters,blood routine,biochemical indicators and organ index measurement were observed.Result: Epimedium extract treatments at 1,3,5 or 10 times of the clinically recommended dose(0.1%)for 7 days had no significant difference on the rate of body weight gain(WGR),specific growth rate(SGR)and condition factor(CF)of Cherax quadricarinatus compared with the blank control group(P > 0.05).The cells in blood of the animals treated with Epimedium extract at 1 time and 3 times of the clinically recommended dose were increased significantly compared with the blank control group(P < 0.01).Epimedium extract treatment at 1 time dose decreased significantly the percentage of large granule cells in the blood of the animals compared with the blank control group(P < 0.05).Epimedium extract had no significant difference on the percentage of small granule cells and transparent cells in the blood of the animals compared with the blank control group(P > 0.05).Epimedium extract treatments at 1,3,5 or10 times of the clinically recommended dose(0.1%)for 7 days significantly decreased the activities of alanine aminotransferase compared with the blank control group(P < 0.01),significantly increased levels of total protein in blood of the animals compared with the blank control group(P < 0.01).Epimedium extract treatments at 1 time and 3 times of the clinically recommended dose had no significant difference on other biochemical values and hepatopancreas indexes compared with the blank control group(P > 0.05),no effect histological structure of hepatopancreas of Cherax quadricarinatu.Conclusion: Epimedium extract is safe for the target animal(Cherax quadricarinatu),when it is used at dose from 1 to 3 times of the clinically recommended dose for 7 consecutive days in clinical application.5.Experimental clinical research of Epimedium extract in Cherax quadricarinatusMethod: 150 healthy and uniform weight Cherax quadricarinatus were randomly divided into five groups,including low dose group of Epimedium extract(feed supplement at 0.1%),medium dose group(feed supplement at0.2%)and high dose group(feed supplement at 0.4%),drug control group and blank control group.Each group consisted of 3 parallels,10 tails each parallel,and the animals was housed in separate boxes.After 7 days of treatments,morphological parameters,blood routine,biochemical indicators,organ index and immune index measurement.Result: The results showed that Epimedium extract significantly increased blood cells in Cherax quadricarinatus compared with the blank control group(P < 0.01),but had no significant difference on the percentage of large granule cells,small granule cells and transparent cells in the blood of the animals compared with the blank control group(P > 0.05).Epimedium extract treatments significantly decreased the activities of glutamic oxaloacetic transaminase and alanine aminotransferase compared with the blank control group(P < 0.01),significantly increased levels of total protein in blood of the animals compared with the blank control group(P < 0.05).Epimedium extract treatments had no significant difference on growth properties and hepatopancreas index and ovarian index of Cherax quadricarinatu compared with the blank control group(P > 0.05).Epimedium extract treatments significantly raised activities of phenoloxidase,superoxide dismutase and lysozyme and significantly increased the levels of glutathione and total antioxidant capacity in hepatopancreas of the animals compared with the blank control group(P <0.05).Conclusion: experimental clinical trial results showed that the clinical recommended dose of Epimedium extract for Cherax quadricarinatus is: 1gram per kg of feed(0.1%),mixed feeding for 7 days. |
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