Preparation Of Decoquinate Nanoliposomes And Verification Of Anticoccidial Effect

Chicken coccidiosis is a kind of disease mainly caused by Eimeria coccidiosis,which mainly occurs in chicks.Due to the damage of the digestive tract after the onset of Chicken,decreased food intake leads to reduced production performance,wasting and even death,which brings serious harm to the poultry industry.Coccidiosis drug resistance brings new challenges to coccidiosis control.Decoquinate(DQ)is a quinoline chemical anticoccidial drug used to prevent coccidiosis in chicken digestive tract,it has the advantages of efficient and safe,but at the same time,DQ is extremely difficult to dissolve in water,and it has shortcomings such as low absorption and utilization.Therefore,it has important clinical significance to make it into water-soluble nanoliposomes.1 Preparation of DQNLFirst,find the right solvent to fully dissolve DQ and materials,we compared a variety of solvents:ethanol,chlorobutane,ethyl acetate,methanol,chloroform,etc.,and finally determined to use chloroform:methanol(1:1)as the dissolving agent.Due to the strong lipophophilic and hydrophobic physicochemical properties of the drug,thin-film dispersion-ultrasonic method was adopted in the preparation of nano-liposomes.Then,the preparation conditions of DQNL were optimized.During the test,the content of DQ was detected by HPLC.Through the absorption peaks of 8 concentration gradient DQ standards,the standard curve equation Y=132653x-60368,R2=0.9997 was established,and its methodology meets the relevant requirements.Then,a single factor test was carried out,5 gradient factors were designed for the lipid ratio,membrane material ratio,water bath temperature and ultrasonic time respectively.The factors with greater influence on the index were screened out by taking the encapsulation rate and drug load as indicators.According to the results of single factor experiment,Minitab was used to design an orthogonal optimization experiment with four factors and three levels of L9(34).The optimum preparation conditions of DQNL were as follows:egg yolk lecithin:drug ratio(w/w)was 10:1;egg yolk lecithin:cholesterol(w/w)ratio of 5:1;the rotary evaporation temperature is 50?;the duration of ultrasound is 15 minutes.2 Characterization of DQNLOn this basis,the optimized method of DQNL prepared was verified,and the structure,particle size and stability were characterized:the average encapsulation rate of DQNL prepared under optimized conditions reached 98.94%,and the drug loading amount reached 6.91%.The transmission electron microscope was used to observe the microscopic morphology of DQNL.The results showed that DQNL had the characteristic structure of liposomes.The results of nanoparticle size analyzer showed that the average particle size of DQNL was 115.6 nm,the polydispersity index was 0.175,and the Zeta potential was-39.1 mV,indicating that the particle size of DQNL reaches the nanometer level,the distribution is uniform and the particles are stable.Then,the DQNL is placed in a 4? environment and sampled every 7 days for particle size analysis.After 28 days of test results,the particle size of DQNL has only increased by 1.9 nm,and the absolute value of Zeta potential was reduced by 3.9 mV,indicating that DQNL has high stability when stored at 4?.3 Determination of the effect of DQNL on E.tenella infectionA drug resistant strain(NT strain)of laboratory coccidiosis strains was selected for testing.According to their body weight,84 12-day-old chicks were divided into blank control group(Blank Control,Con),coccidian-infected group(E.tenella infected,El),decoquinate(EI+40 mg/kg DQ,DQ40),DQNL high dose group(EI+20 mg/L DQNL,DQNL20),DQNL medium dose group(EI+10 mg/L DQNL,DQNL10),and DQNL low dose group(El+5 mg/L DQNL,DQNL5),diclazuril control group(El+2 ?L/L Die,Dic2 consisted of 7 groups with 12 animals in each group.The chicks were inoculated with coccidian oocyst for 3 days,and then kept for a week.During this period,the mental state and feces of the chickens were closely observed.Weighing and sampling on the eighth day,the anticoccidial effect was evaluated by fecal score,relative weight gain rate,cecum lesion score,oocyst value,and anticoccidial index(ACI).The results show that DQNL5 is equivalent to DQ40,indicating that DQNL significantly improves the effect of DQ.It also can soluble in water.After that,in vitro experiments were carried out,DQNL was added into fresh unsporulated oocysts and divided into 9 dose groups.Under uniform conditions,oocyst sporulation was performed,and the sporulation rate was calculated after 72 h.The results showed that 5 mg/L DQNL can significantly reduce the formation of sporulated oocysts,and showed a significant dose dependence4 DQNL freeze-drying technologyConvenient for further DQNL storage and use,we conducted DQNL preliminary discussion of freeze drying process,adding the same concentration of DQNL different types of freeze-dried protective agent,such as sugars,salts,acids,polymers,etc.,through the appearance after freeze drying,the speed of redispersion of the solution after reconstitution and particle size,the protective effect of various freeze-drying protective agents on DQNL was investigated.The results showed that the sugar has obvious protective effects on DQNL during lyophilization,which convenient for stockpile and research.Conclusion:The average particle size of DQNL prepared by the thin-film dispersion-ultrasonic method is 115.6 nm,the particle size is small,and the distribution is uniform.It is miscible with water,and its anti-coccidial effect is significantly stronger than that of the original DQ powder.Its still stable stored at 4? under dark conditions for 28 d,carbohydrate freeze-drying protective agent has obvious freeze-drying protective effect on DQNL.