Screening Of Natural Active Constituents For Liver Protection And Optimization Of Preparation Technology

XM is a natural product,which has a wealth of nutritional value,fast growth and hence the development of its medicinal value has a good application prospects.In previous studies,XM has shown a series of pharmacological effects including regulation of blood pressure,anti-oxidation,immunoregulation,anti-tumor and so on.In the present study,we observed for the first time that XM has a significant hepatoprotective effect as no report on the screening of the active constituents from XM and its preparation technology has been documented before.In this study,the screening of the active constituents from XM was through the classical system solvent and the macroporous adsorption resin methods.The thin film dispersion method was used to prepare the active constituents / phospholipid / sodium cholate / povidone loaded ternary nano-micelle system for solving the problem of solubility and improving the hepatoprotective effect,as well as providing important data for future development and application of XM.Chapter?ReviewThis chapter consists of two parts and provides a review on related literature to this study.The first part introduces the research progress of liver injury,and focus on four aspects of liver function,liver injury,common liver injury model and studies of traditional Chinese medicine on hepatoprotection.The second part comprises the research progress of micelle including the properties of micelle,the classification of micelle,the preparation of micelle and its applications in pharmacy.Additionally,these two parts of the research progress of liver injury and micelles were aimed at providing theoretical basis for the progress of subsequent related studies.Chapter ? Preliminary hepatoprotective effect study on the active fraction of XMIn this chapter,the CCl₄ induced acute liver injury model in mice was established in vivo.The model was fast and stable,and no mouse death occurred during the experiment.The hepatoprotective activities of plant oil extract alongside the water extract of oil residue of XM were studied.The results showed that the water extract of oil residue of XM exhibited better hepatoprotective effect than the plant oil extract.Subsequently,the water extract of oil residue of XM was extracted via the classical system solvent method and the components were separated based on the polarity of the solvents used.The extraction of the active constituent with ether,methylene chloride,ethyl acetate,n-butanol together with water phase were studied and their hepatoprotective activities tested in vivo using CCl₄ treated mice model.The result of the initial screening indicated that n-butanol fraction contained the most effective hepatoprotective agent of XM.The 10%,30%,50%,70% and 90% ethanol fractions were isolated from the n-butanol part through macroporous adsorption resin method.The hepatoprotective effects of 10%,30%,50%,70% and 90% ethanol fractions were tested in mice.The results depicted that the optimal hepatoprotective active part of XM was in the 30% ethanol portion.Again,the study of five different concentrations of ethanol in vitro using bu cell still clearly showed that 30% ethanol fraction contains the optimal hepatoprotective active part of XM.Chapter ? Component analysis of 30% ethanol active fraction and preformulation studiesIn this chapter,the qualitative analysis of 30% ethanol active fraction was carried out.The Saponins,flavonoids,alkaloids and polysaccharides of 30% ethanol active fraction were qualitatively detected in two different methods.The results showed that the main component of 30% ethanol active fraction is flavonoids and other ingredients that have no obvious phenomenon.UV Spectrophotometry was established as the analytical method for studying the 30% ethanol active fraction in vitro.The established method provided good linear relation in the range of 4.64-46.4 ?g·m L-1,and the intra-day and inter-day precision was high?RSD <2%?.In addition,the 30% ethanol hepatoprotective active part solution remained stable within 72 h,and its total flavonoids content was 61.56%.The results of the equilibrium solubility declared that the solubility of the 30% ethanol active fraction in p H 1.2?maintained using HCl?water was?346.03 ± 40.34??g·m L-1,and this was highest at p H 7.2?maintained using PBS??478.42 ± 42.08??g·m L-1.These results showed that the 30% ethanol active portion was insoluble in the three kinds of media.Meanwhile,it also provided the basis for the confirmation of sink condition in the next study of the 30% ethanol active fraction loaded ternary nano-micelle.Chapter ? Preparation and in vivo evaluation of 30% ethanol active fraction loaded ternary nano-micellesIn this chapter,30% ethanol active fraction loaded ternary nano-micelles were prepared through thin film dispersion method.The average particle size,encapsulation efficiency and apparent clarity were taken as the evaluative criteria.The optimal formulation technique was conducted using single factor experiment and orthogonal test optimization.The final optimized results of 30% ethanol active fraction loaded ternary nano-micelles were consisted of 80 mg dosage,400 mg phospholipids,300 mg sodium cholate,and 300 mg PVP K30.The results of evaluation of optimal formulation in vitro showed that 30% ethanol active site loaded ternary nano-micelles was clear and transparent with homogenous distribution.The average particle size was 46.38±1.78 nm,polydispersity index of 0.186 ± 0.08,Zeta potential was-23.67 ± 1.21 m V and the encapsulation efficiency determined to be 86.38 ± 1.46 %.The results of preliminary stability test showed that 30% ethanol active fraction loaded ternary nano-micelles had good dynamic stability at 60 oC for at least 1 month.In vitro drug release studies showed that the cumulative release rate alongside release rate of 30% ethanol active fraction loaded ternary nano-micelles were significantly higher than those in 30% ethanol active portion.The cumulative release of 30% ethanol active portion was only about 55% within 72 h whiles that of 30% ethanol active fraction loaded ternary nano-micelles was more than 95% within72 h,indicating that the ternary nano-micelles could be used as the carrier for the XM drug.Hence,it could improve the solubilization of 30% ethanol part coupled with greater enhancement of the drug release ability.Chapter ? Pharmacodynamics study of 30% ethanol active fraction ternary nano-micelles on CCl₄ induced hepatic injuryIn this chapter,the CCl₄ induced acute liver injury model in vivo was established using mice.The commercially available products were used as positive control group.The 30% ethanol active portion and 30% ethanol active fraction loaded ternary nano-micelles were administered via intragastric administration for one week.The results indicated that all the evaluation indexes of 30% ethanol active fraction loaded ternary nano-micelles group and 30% ethanol active counterpart were better than the positive control group.Moreover,the hepatoprotective effect of 30% ethanol active fraction loaded ternary nano-micelles group was highly significant than the 30% ethanol portion.The 30% ethanol active fraction loaded ternary nano-micelles showed ideal hepatoprotective effect in terms of same dosage.The results depicted that 30% ethanol active portion prepared through ternary nano-micelles technology improved its hepatoprotective activity.Furthermore,the results indirectly posited that the ternary nano-micelles technology could enhance the absorption and oral bioavailability of 30% ethanol active fraction in vivo,and also exhibited potential value in clinical application.