Construction And In Vitro And In Vivo Evaluation Of Formononetin Self-microemulsion Delivery System

Self-microemulsion drug delivery system is an oil-like mixture composed of drugs,oil phase,emulsification and co-emulsification,which can spontaneously form highly dispersed microemulsions under the peristalsis of the gastrointestinal tract.It has good stability and high dispersion of drugs,and is especially suitable for insoluble natural active ingredients to improve water solubility and bioavailability.Formononetin is a kind of isoflavone compound with excellent pharmacological activity,but its application also has the following limitations,including insoluble in water,low bioavailability,unknown absorption mechanism,and lack of related studies on increasing solubility and promoting absorption.In this study,formononetin was used as model drug to construct its self-microemulsion drug delivery system.In the study,it was found that its solubility in the excipients of self-microemulsion was not high,which led to its low drug loading in the self-microemulsion drug delivery system,which limited its application.In order to improve the drug loading of formononetin in the self-microemulsion drug delivery system,this study applied the nanocrystalline technology in the self-microemulsion drug delivery system through the analysis of physicochemical properties of formononetin and the review of relevant literature.Prescription screening,preparation process optimization,property characterization,cell safety evaluation,cell uptake evaluation,oral absorption and bioavailability evaluation of formononetin nanocrystalline drug delivery system and its self-emulsion drug delivery system were conducted.The full text is mainly divided into the following six chapters:Chapter one:ReviewsIn this chapter,the physical and chemical properties,pharmacological effects and related preparations of formononetin are reviewed.The characteristics,preparation methods,characterization and evaluation of nanocrystalline drug delivery systems and their research progress in the field of pharmacy were introduced.In addition,the characteristics,composition,characterization and evaluation of self-microemulsion drug delivery system,as well as related research progress at home and abroad were also described.Finally,the purpose and content of this study are put forward.This chapter lays the foundation for the study of formononetin nanocrystalline and its self-microemulsion delivery system in the later stage.Chapter two:Preformulation studiesIn this chapter,the in vitro HPLC analysis method of formononetin was established,and the methodological verification of the method was carried out,which met the requirements of methodological verification.The equilibrium solubility and oil-water partition coefficient（Log P）of formononetin in different media were measured.The saturation solubility of formononetin in each medium was less than 2μg/m L,and the Log P value in each medium was between 1-2.By polarizing light microscope and powder X-ray diffraction analysis,the results showed that formononetin crystallized in bulk and had a strong crystallization peak.Subsequent crystallization inhibition experiments were conducted to preliminarily screen out the excipients with better crystallization inhibition ability of formononetin.The above research results before prescription laid a theoretical foundation for the preparation of formononetin nanocrystals and formononetin nanocrystals load self-microemulsifying drug delivery system.Chapter three:Construction and in vitro evaluation of the drug delivery system of formononetin nanocrystalsIn this chapter,formononetin nanocrystals were prepared by reverse solvent precipitate-probe ultrasonic method with formononetin nanocrystals average particle size and PDI as the main investigation indexes.First,single-carrier formononetin nanocrystals were used for preliminary screening to determine the preparation process,and then the composite stabilizer formononetin nanocrystals were further screened.Finally,the optimal formulation and preparation process of formononetin were selected.Subsequently,the appearance,particle size distribution,electron microscope morphology,drug loading,ATR-FTIR,DSC,PXRD and stability of the nanocrystalline drug delivery system were investigated,as well as the drug release in vitro.The prepared formononetin nanoparticles had a grain size of（302.19±5.02）nm,a PDI of0.193±0.015,Zeta potential of（-21.22±0.91）m V and drug loading of（60.82±0.12）%.The results of ATR-FTIR,DSC and PXRD showed that the nanocrystals of formononetin existed in amorphous state and polycrystalline state composed of crystalline state.The stability experiments show that the composite stabilizer has better stability than the monononetin nanocrystals prepared by single stabilizer.The equilibrium solubility and oil-water partition coefficient of formononetin nanocristal in different media were greatly improved compared with the bulk drug.The cumulative release rates of formononetin nanocrystalline in p H 1.2 HCl solution,p H 6.8PBS and distilled water were（50.74±2.10）%,（43.06±1.60）%and（49.36±2.20）%,respectively.Chapter 4:Construction and in vitro evaluation of formononetin nanocrystals load Self-microemulsifying drug delivery systemIn this chapter,formononetin nanocrystals load self-microemulsifying drug delivery system were prepared using formononetin nanocrystals as model drugs.Based on the equilibrium solubility of formononetin nanocrystals in different excipients,the appropriate oil phase,emulsifier and coemulsifier were selected by compatibility test and pseudo-ternary phase diagram was drawn by titration method,which were Capmul^?MCM C8 EP/NF,TW 20,PEG 200,respectively.The optimum prescription and its preparation process were selected by single factor experiment.The characteristics of the optimal prescription in vitro were preliminarily investigated.The formononetin nanocrystals load self-microemulsifying drug delivery system was transparent light-yellow liquid at room temperature,and the self-microemulsion solution obtained after dilution was homogeneous light-yellow transparent liquid.The microemulsion showed spheroid shape with uniform size and uniform distribution without adhesion phenomenon.The mean particle size,PDI and Zeta potential were（20.65±1.42）nm,0.130±0.015 and（-26.57±0.35）m V,respectively.The drug loading was（19.371±0.037）mg/g and the encapsulation rate was（96.85±0.08）%.The cumulative release rates of formononetin nanocrystalline and its self-microemulsion delivery system in p H 1.2 HCl solution,p H 6.8 PBS and distilled water were（95.23±4.65）%,（83.06±4.29）%and（90.00±3.91）%,respectively.Chapter 5 Cell safety evaluation and cell uptake of formononetin nanocrystalline and formononetin nanocrystals load self-microemulsifying drug delivery systemIn this chapter,HPLC analysis method was first established for the determination of intracellular drug concentration.By using Caco-2 cells,the cell safety of formononetin,formononetin nanocrystals,formononetin nanocrystals load self-microemulsifying drug delivery system and coumarin-6 were evaluated to verify their safety and investigate the safe concentration of coumarin-6.It is convenient for subsequent cell uptake experiments.IC₅₀ values were fitted by Graph Pad Prism,and formononetin apis were obtained.The IC₅₀ values of formononetin nanocrystals and self-emulsified formononetin nanocrystals were（42.13±1.52）μg/m L,（32.22±1.41）μg/m L and（70.20±3.43）μg/m L,respectively,indicating good safety.Qualitative and quantitative studies on cell uptake were carried out.Coumarin-6 was used as fluorescent dye to carry out qualitative study on cell uptake of the preparation.The qualitative results showed that the fluorescence signal intensity was self-microemulsified coumarin-6 nanocrystals load self-microemulsifying drug delivery system>coumarin-6 nanocrystals>coumarin-6,indicating that drug preparation into nanocrystals can promote drug uptake by cells,and preparation into self-microemulsified nanocrystals can further increase drug uptake by cells.The uptake time and temperature were determined by quantitative analysis.At the same time,it was found that the uptake rate of cells was in the order of formononetin nanocrystals load self-microemulsifying drug delivery system>formononetin nanocrystles>formononetin.Therefore,the preparation of formononetin into nanocrystalline and the formononetin nanocrystals load self-microemulsifying drug delivery system can effectively increase the drug uptake capacity.Chapter 6 Studies on intestinal absorption and bioavailability of formononetin nanocrystals and formononetin nanocrystals load self-microemulsifyingIn this chapter,HPLC analysis method of formononetin was established in vivo,and the absorption and oral bioavailability of formononetin,formononetin nanocrystals and formononetin nanocrystals load self-microemulsifying drug delivery system in rat models were investigated.The results showed that formononetin was absorbed in each segment of small intestine,and with the change of concentration.The absorption rate constant Ka and absorption percentage（%）were almost unchanged.The experimental results showed that formononetin concentration may be passive diffusion in the range of 2-20μg/m L.Formononetin nanocrystals and formononetin nanocrystals load self-microemulsifying drug delivery system can effectively improve the absorption rate and degree of formononetin in each intestinal segment,and the improvement degree of formononetin nanocrystals load self-microemulsifying drug delivery system is more obvious,which is conducive to improve the oral absorption of formononetin.Oral bioavailability results of rats showed that both formononetin nanocrystalline and formononetin nanocrystals load self-microemulsifying drug delivery system could improve the bioavailability of formononetin.Relative bioavailability of formononetin nanocrystalline was 154.80%compared with formononetin bulk drug.The relative bioavailability of formononetin nanocrystals load self-microemulsifying drug delivery system reached 557.73%.