Optimal Design Of In Situ Gel Sustained Release Agent For Melation And Evaluation In Vivo Of Broilers

The biological effects of melatonin are extensive,including anti-inflammatory,antioxidant,and immune regulation.However,melatonin has low bioavailability because it is metabolized quickly in animals,which is particularly important for the development of melatonin slow preparations.First,an analytical method was established to determine the melatonin content by high performance liquid chromatography.Second,the formulation and process were optimized by single factor and Box-Behnken response surface design method,and pharmacokinetics of melatonin temperature-sensitive gel in situ was researched,through the drawing of the standard curve,the determination of precision and recovery rate.Last,the antioxidative effect was tested through in vivo experiments in heat-stressed broilers,and the thermosensitive in situ coagulation of melatonin was investigated through high temperature test,low temperature test,accelerated test and long-term test.The quality of the glue provides a theoretical basis for the clinical application of melatonin sustained-release preparations.Experiment 1,Established a temperature-sensitive in situ gel analysis method for melatonin: The content of melatonin were determined by high performance liquid chromatography.The results indicate that,there was a good linear relationship between peak area and concentration of melatonin.The standard curve equation is Y=57.988x-9.9134,R2=0.9997,when the concentration of melatonin is between 0.5~100μg/m L.Experiment 2,The thermosensitive in situ gel analysis method for melatonin: In order to screen the best melatonin temperature-sensitive in situ gel process,and lay a foundation for further animal test.The appearance,gelation temperature,gelation time and release rate of temperature-sensitive in-situ gel of melatonin were comprehensively evaluated,which was synthesized by single factor and Box-Behnken design method.The results showed as follows:(1)The optimal formula of melatonin temperature sensitive in-situ gel was obtained by response surface methodology:0.15% melatonin,22% P407,1.5% P188,0.4% PEG4000,0.5% HPMC(15KM),and water for injection 75.45%.(2)By optimizing the temperature-sensitive in situ gel process of melatonin,it was found that the release time of the optimal scheme could reach five days,which obviously played a slow release effect.Experiment 3,Pharmacokinetics and antioxidant effect of temperature-sensitive in situ gel of melatonin: Sustained release effects were compared by analyzed pharmacokinetic parameters of the original solution and gel.And antioxidant effect was validation in vivo test of heat stressed broilers.The results show that:(1)The peak time of the original solution was 0.5 h,and the peak time of the gel in broilers was 72 h.(2)In the liquid group and gel group,the average retention time was the longest,and the peak concentrations with the best effect were 890.22 μg/ m L and 561.66 μg/ ml,respectively.The curve areas were 890.70 h*μg/ ml and 399068.75 h*μg/ ml,respectively,indicating that the bioavailability of gel group was higher than that of raw solution group.(3)Antioxidant performance observation: on the first day,the contents of GSH-PX,MDA,SOD,T-AOC and CAT in gel group and raw liquid group were significantly better than those in control group(P<0.01),but there was no significant difference between the two groups(P>0.05),indicating that both groups had extremely significant antioxidant effect.On the third and fifth days,the contents of GSH-Px,MDA,SOD,T-AOC and CAT in gel group were significantly better than those in control group(P<0.05),while there was no significant difference between the original liquid group and control group(P>0.05).In terms of serum antioxidant effect of broilers,the in situ gel slow-release effect of melatonin could reach five days.The results showed that melatonin gel had better antioxidant effect.Experiment 4: The temperature sensitive in situ gel of melatonin was determined by high temperature test,low temperature test,accelerated test and long-term test to examined the quality of melatonin content temperature-sensitive in the situ gel.The results show that:(1)The gelation effect in vivo and in vitro is obvious: When injected into the animal,the gel preparation can produce rapid gelation within 1 min.The gel is colorless translucent gel solution under storage conditions,and becomes semi-solid gel after reaching the required gelation temperature.(2)Preliminary stability studies indicate that the preparation needs to be stored at low temperature(1~4℃)under dark conditions.In conclusion,the time required for the release of melatonin temperature-sensitive in-situ gel injection prepared in the experiment can reach five days,and the preparation method is simple and reliable,with stable quality,which can significantly reduce The Times of medication,and has good application value.