Optimization Of Preparation For Methanol Extraction Of Eupatorium Adenophorum Spreng And Its Acaricidal Activity On Dermanyssus Gallinae

In recent years,people have gradually begun to pursue quality and safety of chickens with excellent flavor and outside feeding under the forest or stocking in the mountain.However,the intensive,mountain-breeding farming model is extremely benefitial to the infection of chicken body surface parasites.The common surface parasites of chickens include louse,mite,flea,tick,etc.Once they break out,the harm to the chicken industry is no less than other diseases.At present,food safety has been paid more and more attention,and commonly useful insecticides of chemical synthesis are limited.Therefore,it is necessary to seek for safe,effective and environment-friendly drugs which does not affect the quality of chicken meat.Phyto-drugs are natural active substances with the advantages of no residue,low toxicity,and wide resources.In this experiment,The methanol extracts of Eupatorium adenophorum as the research object was optimized by orthogonal test,and the content of its main ingredient named as 9-carbonyl-10,11-dehydrozeolidone was detected by HPLC.To study the acarecidal effect of different concentrations of methanol extracts on chicken mites in vitro,and to provide references for the rational development and use of Eupatorium adenophorum as a valuable resource,we have designed this experiment.1.Optimization of methanol extract from Eupatorium adenophorumThe L9（3⁴）orthogonal table was used to optimize the experimental design.The extraction rate of methanol extract of Eupatorium adenophorum was used as an index to examine the effect of methanol concentration（A）,soaking time（B）,soaking number of times（C）,ratio of solid to liquid（D）on the extraction rate.The results showed that the highest extraction rate of Eupatorium adenophorum methanol extract was A₃B₃C₂D₁,the maximum extraction rate was obtained when the re-methanol concentration was 80%,the soaking time was 48 h,soaking 3 times,and the solid-liquid ratio was 1:4.According to the results,we confirmed that the B（time）factor has little effect on the test results,so the order of each factor is:C>A>D>B,that is the soaking number of times>methanol concentration>solid-liquid ratio>soaking time.2.HPLC analysis of Euptox A contentIn this experiment,the content of Euptox A was detected by high performance liquid chromatography in the methanol extract of Eupatorium adenophorum obtained by orthogonal test.The results showed that the Euptox A peak time was:12.338 min.By area normalization method,the content of Euptox A in Eupatorium adenophorum methanol extract was determined.We found that the extraction method was A₂B₂C₃D₁with methanol concentration of 70%,soaking time of 36h,soaking 4 times,and the ratio of solid to liquid of 1:4.It has the highest content of Euptox A.Analysis of variance revealed that there were no significant differences between the 9 different extraction methods.3.Acaricidal activity of methanol extraction on Dermanyssus gallinaeIn this study,methanol extraction of Eupatorium adenophorum A₂B₂C₃D₁ was used as a solvent and 10%Tween-80 was used as a solvent to prepare 4 mg/mL,8mg/mL,16 mg/mL low,medium and high concentrations.The drug solution,with 2%ivermectin as a positive control group and 10%Tween-80 as a solvent control group,was used to study the acarecidal effect of methanol extract of Eupatorium adenophorum on Dermanyssus gallinae in vitro.The results showed that the killing rate of Eupatorium adenophorum methanol extract at 4 mg/mL,8 mg/mL and 16mg/mL was significantly different from that of the solvent control group,and it was positive at 1h.The group effect was significantly higher than that of each concentration group,and the killing rate of the Dermanyssus gallinae reached 50%at a concentration of 16 mg/mL at 1h.At 4h,the positive group had no significant effect compared with each concentration group,and the killing rate of the three-concentration gradient on the Dermanyssus gallinae was more than 60%.At 8h,there was no significant difference between the positive group and each concentration group,and the killing rate of the Dermanyssus gallinae reached 100%after 8h at a concentration of 16 mg/mL.At the same time,the killing rate increased with the concentration of methanol extracts in vitro.The CLL model was used to analyze the toxicity of methanol extract of Eupatorium adenophorum on the Dermanyssus gallinae in vitro.The results showed that the methanol extract of Eupatorium adenophorum was at 4 mg/mL,8 mg/mL and16 mg/mL for Dermanyssus gallinae.The LT50 was 2.096h,1.444h,and 1.119h,respectively.As the concentration increased,the value of LT50 gradually decreased.The LC50 at 1 h,4h and 8h were 15.498mg/mL,2.269mg/mL and 0.732mg/mL,respectively.The results showed that the methanol extract of Eupatorium adenophorum had strong acaricidal activity on Dermanyssus gallinae in vitro.All in all,Eupatorium adenophorum has the potential to develop a new medicine for killing Dermanyssus gallinae.