Study On The Preparation,in Vivo And In Vitro Absorption Of Chrysin Self-microemulsified Preparation And Its Anti-hyperuricemia Effect

Objective:Chrysin(5,7-dihydroxyflavone,chrysin,Chr),as a brass chemical component of traditional Chinese medicine,has many biological effects such as anti-oxidation,anti-inflammatory,antibacterial and anti-virus,but because of its Water insolubility leads to poor absorption in the body and rapid glycosylation and metabolism of the 5 and 7 hydroxyl groups in the body,resulting in lower activity,which limits the clinical application of the drug.Self-microemulsion drug delivery system(SMEDDS)is a nanometer drug delivery system that has been studied in recent years.It can improve the solubility of poorly soluble drugs,increase the lymphatic transport of drugs,improve oral bioavailability and stability,it can broaden the population of drug users,and it's easy to produce on a large scale,providing a new route of administration for the oral absorption of a large number of poorly water-soluble chemical drugs and active ingredients of traditional Chinese medicine.In this study,We researched the physical and chemical properties,preparation and characterization,in vivo and in vitro absorption and in vivo pharmacodynamics of the self-microemulsified drug delivery system of chrysin(Chr-SMEDDS),and analyzed its oral absorption mechanism from the cellular and molecular levels,aiming to improve its solubility and drug absorption,providing research foundation for the development and clinical application of chrysin's new oral preparations,and provide experimental basis for the research and development of SMEDDS.Method:1.Establishment of Chr in vitro assay and analysis method;Shaker method was used to determine the equilibrium solubility of Chr in different medias;The classic shake flask method was used to determine the oil-water partition coefficient of Chr;Ultraviolet spectrophotometry was used to determine the dissociation constant of Chr,as to provides a theoretical basis for the follow-up Chr-SMEDDS formulation research.2.With the formation of self-microemulsion time as the evaluation index,the single factor test method was used to investigate the influence of stirring speed,temperature,dilution medium and dilution factor on the preparation of SMEDDS respectively;according to the measurement results of the saturation solubility of Chr in different media,the Prepare SMEDDS with oils,surfactants and co-surfactants with greater drug solubility and lower toxicity,and conduct phase separation experimental studies to screen out stable compatibility for prescription research;pseudo-ternary phase diagram method was used for prescription screening,The star point design-response surface method was used to optimize the prescription;according to the maximum drug loading,the state of microemulsion formation,self-microemulsion time and particle size are used as indicators to investigate its influence on the formation of SMEDDS,and fine-tune the prescription according to the experimental results.Determined the final drug loading.3.According to observe the color,consistency and fluidity of the blank SMEDDS and Chr-SMEDDS of the prescription amount,the appearance was evaluated.After adding the ultrapure water to the prescription amount of blank SMEDDS and Chr-SMEDDS for complete emulsification,the p H value was measured with a precision p H meter,the particle size and Zeta potential were measured by a Malvern laser particle size analyzer,and their shape was observed under a transmission electron microscope and Dyeing method was used to identify the type of microemulsion;With the content change of Chr as an index,low temperature,high temperature,cold and heat cycle and sample retention test were used to investigate the stability of Chr-SMEDDS;Centrifugal method was used to determine the encapsulation efficiency of Chr-SMEDDS;The overall liquid balance reverse dialysis method was used to study the in vitro release rate,and the in vitro release behavior was fitted with a simulation equation.4.Taking SD rats as the test object,the LC-MS/MS method was used as the detection method,With the chrysin suspension as the reference preparation,after oral administration of Chr-Suspension and Chr-SMEDDS and the emulsified Chr-SMEDDS was injected intravenously,the blood drug concentrations at different time points in the rat were measured,and the average blood drug concentration-time data was analyzed by DAS 2.0software,non-compartmental model for statistical moment fitting analysis.5.To establish an in vitro absorption model of Caco-2 cell monolayer,and evaluate the model by microscopic observation,TEER determination and alkaline phosphatase activity detection;CCK-8 method was used to determine the survival of Caco-2 cells with different concentrations of Chr's preparations;To investigate the effects of different time,drug concentration and P-gp inhibitors on cell uptake and cell transport;Western blot and immunofluorescence were used to detect the expression of ZO-1 and Occludin proteins in different Chr's preparations,and to preliminarily explore the mechanism of SMEDDS which affecting the oral bioavailability of Chr.6.Using Oxonic aid potassium salt(OA)to induce the establishment of a mouse hyperuricemia model,and according to measure the changes in serum UA and XOD levels,determine whether Chr-SMEDDS is effective in hyperuricemia mice and possible mechanism of action.Result:1.Established an HPLC in vitro content determination method for Chr,In the methodological investigation,the linearity,precision,repeatability,solution stability and sample recovery experiments all meet the requirements;the equilibrium solubility of Chr in water is 38.43?g·L^-1,and within the range of p H 1.2-11,it increases with the increase of p H.In the selected dissolution medium,the equilibrium solubility of Chr in diethylene glycol monoethyl ether(transcutol HP)is the largest,reaching 6.63 g·L^-1;the oil-water partition coefficient(lg P)of Chr in different p H solutions is determined,The results showed that at p H 1.2-9,the lg P of chrysin in different p H solutions was between 4.34 and 4.41,with no significant change.When the p H reached 10,the lg P of chrysin suddenly decreased.When the p H reached 11,The lg P of chrysin is further reduced;chrysin has two dissociation constants measured by ultraviolet spectrophotometry,namely p K_a1and p K_a2,the value are 6.52 and7.92 respectively,which are weakly acidic drugs.2.Through the single factor test method,with the formation of self-microemulsion time as the evaluation index,the influence of stirring speed,temperature,dilution medium and dilution factor on the preparation of SMEDDS were investigated respectively,and 50r/min was selected as the finally stirring speed,37?as the finally ambient temperature of test,Ultra-pure water as the finally dilution medium,200 times as the finally dilution factor;according to the results of the phase separation test,combined with the results of the preliminary experiments,selecting MCT,Ethyl Oleate,Oleic acid and a mixture of MCT and Oleic acid as the oil phase,Kolliphor ELP and cremophor RH40 as a surfactant,Transcutol HP is used as a co-surfactant for subsequent formulation studies;a mixture of MCT and Oleic acid(mixing ratio of 1:1)was screened out by using the pseudo-ternary phase diagram method as the oil phase,and cremophor RH40 was used as the surfactant,Choosing Km(1:1)as the mixing ratio of surfactant and co-surfactant.Using the star-point design-response surface method to optimize the preparation of SMEDDS,the best formula for preparing SMEDDS was the mixed oil phase of MCT and Oleic acid(mixing ratio of 1:1 respectively)24%,cremophor RH40 32%,and Transcutol HP 44%.The optimal formulation was verified,and the results showed that the star point design-effect surface optimization had a good predictive effect;The prescription determined that the theoretical drug loading amount of SMEDDS for Chr was 12.45 mg/g.Taking into account the stability of the entire drug loading system,the actual drug loading amount was 5mg/g.3.Under normal temperature conditions,the blank SMEDDS was a white transparent viscous liquid,and Chr-SMEDDS was light yellow.At 4?,the fluidity of the blank SMEDDS and Chr-SMEDDS decreased without other significant changes.After adding ultrapure water to the Chr-SMEDDS for emulsifying completely,the microemulsion showed a slight yellowish color.Under the black background condition,the formed microemulsion showed light blue opalescence;The Chr-SMEDDS's p H value is 5.6 after diluting with 100 purified water.The particle size and Zeta potential of the blank SMEDDS microemulsion and Chr-SMEDDS microemulsion were measured by Malvern laser particle size analyzer as 22.62 nm and 27.34 nm,respectively.When the Chr-SMEDDS was added with 50,100,and 200 times ultrapure water,the Zeta potential after complete emulsification were-16.5m V,-22.2m V and-27.3m V respectively;Chr-SMEDDS microemulsion was spherical in shape,uniform in size and distribution,with a particle size of20-40nm under a transmission electron microscope.staining method was used to identify the microemulsion formed by Chr-SMEDDS as O/W type;stability and long-term retention sample results showed that Chr-SMEDDS can maintain its original color and clarity for 6 months at room temperature,and there was no obvious change in particle size and drug loading,indicating that Chr-SMEDDS had good stability;the average encapsulation rate of Chr-SMEDDS determined by centrifugation was 91.73%;the release was under investigation,The release of Chr-SMEDDS reached more than 85%within 6 h,while the release of Chr-Suspension at 6 h was close to 55%.After fitting the release curve,it was found that the in vitro release of Chr-SMEDDS conformed to the first-order kinetic equation,while the in vitro release of Chr-suspension conformed to the Higuchi equation.4.The results of in vivo pharmacokinetic studies showed that the half-life(t_1/2z)of Chr-ME intravenous injection was(9.9±3.6)h,and the area under the curve of blood drug concentration and time(AUC_0-t)was(651.0±25.4)?g?h/L.After intragastric administration,the AUC_0-tof Chr-Suspension and Chr-SMEDDS were(84.5±6.0)?g?h/L and(230.4±9.8)?g?h/L,respectively.Compared with Chr-Suspension,the bioavailability of Chr-SMEDDS was increased by 2.73 times.The absolute bioavailability of Chr-Suspension and Chr-SMEDDS relative to Chr-ME were1.48%and 4.05%,respectively.5.Established a Caco-2 cell monolayer model,microscopic observation of tight junctions between cells,clear boundaries,21 days TEER value could reach 696.3?·cm~2,AP side and BL side alkaline phosphatase ratio was 6.25;The CCK-8 method was used to determine the viability of Caco-2 cells in different concentrations of Chr test solution,Chr-SMEDDS test solution and blank SMEDDS test solution.The results showed that when the content of Chr was less than 2.8 mg/L,the Caco-2 cell viability Survival rate exceeds 90%;SMEDDS can significantly increase the uptake and transport of Chr by Caco-2cells under different time of action and drug concentration,while P-gp inhibitors had no significant effect on the uptake and transport of Chr and Chr-SMEDDS;Western blot And immunofluorescence detection results showed that the Chr-SMEDDS and SMEDDS could significantly reduce the expression of ZO-1 and Occludin protein compared with Chr,indicating that SMEDDS could open the tight junction of cells and allow the drug to be transported across the membrane through the cell bypass,increasing Chr's intestinal absorption thereby.6.A mouse hyperuricemia model(HUA)induced by Oxonic aid potassium salt(OA)had been established.At doses of 20 and 40 mg/kg,Chr-Suspension had no significant effect to reduce the mice Serum UA and XOD levels,while Chr-SMEDDS could significantly reduce the serum UA and XOD levels in acute HUA model mice at a dose of 40 mg/kg.Conclusion:In this study,We prepared a self-microemulsified chrysin(Chr-SMEDDS)with high encapsulation efficiency,stable properties and a particle size of less than 100 nm successfully;the SMEDDS prepared could significantly improve the solubility and dissolution of chr,increase its absorption in the body and improve its bioavailability in the body;the mechanism of SMEDDS to promote the absorption of chrysin might be the increasing its dissolution in the body,enhancing the uptake of drugs by cells,and enhancing the transport of drugs by opening tight cell junctions;when a certain dose was reached,Chr-SMEDDS could significantly reduce the uric acid(UA)value of HUA model mice,which might increase the absorption of Chr in vivo with SMEDDS,and inhibit the activity of xanthine oxidase(XOD)thereby.