| Pueraria lobata is the dried root of the leguminous plant Pueraria lobata（Willd.）Ohwi.Puerarin（7,4’-dihydroxyisoflavone-8-glucopyranoside,Puerarin,PUE）,the main component of Pueraria Lobata,has pharmacological activities like lowering blood pressure[1],improving cerebral ischemia-reperfusion injury[2],improving glucose metabolism[3]and anti-cancer[5-6].However,PUE is only clinically available in two dosage forms:injection and eye drops,PUE injection has many adverse reactions[7]and poor patient compliance,while eye drops are mainly used for glaucoma[8].Oral dosage forms are safer and more patient compliant than injections,making them suitable for long-term administration.However,PUE is a class IV drug in BCS classification[9],and its oral bioavailability is low.The research group’s previous study found that daidzein（7,4’-dihydroxyisoflavone,Daidzein,DAZ）in Pueraria lobata can promote the permeability of PUE,Meanwhile,DAZ and its derivatives also have antihypertensive activities[10-11].The two drugs were simultaneously loaded in order to improve the oral bioavailability and efficacy of PUE.F127/HS15 self-assembled micelle system is a hybrid micelle system composed of a non-ionic triblock copolymer（F127）and a non-ionic surfactant（HS15）[12],it can improve the solubility of the drug.However,micelles are thermodynamically and kinetically unstable systems,which are not conducive to storage and consumption.Lyophilized powder,as an intermediate of solid preparation tablets and granules,has the characteristics of high stability and easy to carry.Objective:In order to improve the bioavailability and efficacy of PUE,F127/HS15self-assembled mixed micelle system was used as the carrier to prepare PUE and DAZ co-loaded mixed micelle（PD-M）and PUE and DAZ co-loaded mixed micelle freeze-dried powder（PD-M-LP）,and the pharmacy properties,in vitro release,oral pharmacokinetics and pharmacodynamics of PD-M and PD-M-LP were evaluated.Method:（1）First to establish a HPLC method for determining the methodology of content of PUE,PD-M,which was prepared by thin-film hydration of PUE dosage,dosage of DAZ F127,total amount of carrier,the amount of hydration of PUE the influence of leakage rate,determine the dosage of PUE,dosage of DAZ,ratio of F127the three biggest influence factor,and the Box-Behnken response surface optimization PD-M preparation craft and prescription,finally by means of freeze drying will PD-M curing.The effects of the type and dosage of lyophilized protectant on the appearance of lyophilized powder,PDI and RDI after resolution were investigated.（2）The appearance,particle size,transmission electron microscopy（TEM）,scanning electron microscopy（SEM）,fourier infrared spectroscopy（FTIR）,differential scanning calorimetry（DSC）and hydrogen nuclear magnetic resonance（HNMR）of PD-M and PD-M-LP were studied.The in vitro release of PUE and DAZ in PD-M was studied by dialysis bag method.（3）Taking male SD rats as the research object,the pharmacokinetics of PD-M after oral administration was studied by intragastric administration.HPLC method was established to determine the content of PUE in rat plasma,and internal standard method was used to calculate the plasma concentration of PUE with daidzein as the internal standard.（4）Taking male males SHR and WKY rats as the research object,divided into 7 groups,each group of nine,lavage with rat tail volume measuring method for the continuous monitoring PD-M dose group,high dose group,active pharmaceutical ingredients in the control group,model control group,blank micelle group,positive control group（Losartan Potassium）and normal control group seven groups of rats and ten weeks of systolic pressure,diastolic pressure,mean blood pressure,heart rate and weight.Result:（1）The chromatographic conditions of puerarin were as follows:acetonitrile:0.1%phosphoric acid water=16:84;Finally,the optimized prescription and process were as follows:the dosage of puerarin was 80 mg,the dosage of daidzein was3.50 mg,and the proportion of F127 was 75%.At this time,the leakage rate of puerarin was the lowest.The repeated experiment was conducted according to the optimized prescription,and the results showed no significant difference with the predicted value.4%（w/v）glycine was used as a lyophilized protectant.（2）The pharmacy characterization results showed that PD-M was clear and transparent,while PD-M was in white powder form.The particle size distribution of PD-M and PD-M-LP after resolution is almost the same,which is in the range of 18-20 nm.Transmission electron microscopy showed that PD-M was spherical and had a complete appearance.Scanning electron microscopy showed that the morphology of PD-M-LP was rod-like,lamellar and acicular,the lamellar morphology was dominant.Fourier transform infrared spectrum analysis showed that the infrared spectrum of PD-M-LP was almost the same as that of blank micelles,the characteristic peaks of drugs were almost covered by excipients,indicating that drugs were all contained in PD-M-LP.The DSC analysis showed that the melting point peak of PD-M-LP was almost the same as that of the blank micelle,and there was no characteristic peak of the drug,indicating that the existence form of the drug in PD-M-LP was amorphous.The NMR hydrogen spectra showed that the chemical environment of PD-M hydrogen atoms changed in PD-M,which was different from that in the drug,indicating that the drug was encapsulated in the carrier.In vitro release,according to the results of both the PUE and DAZ in PD-M release curve with the Weibull model,the correlation coefficient R2 were greater than 0.99,but the release curve was not similar to that of similarity factor1)2=33<50,the release rate of DAZ was slower than PUE,indicated that the PUE and DAZ distribution in different areas of the micelle,DAZ tended to be distributed in internal scanty water,the PUE tended to be distributed in external secondary steam water.（3）The pharmacokinetic results showed that the AUC（0→360）,AUC（0→∞）and Cmaxof PD-M were1.21、1.34 and 1.77 times that of PUE,respectively,and the relative bioavailability increased to 121%.（4）Pharmacodynamics results showed that PD-M medium dose group and high dose group had significant effects on the systolic,diastolic and mean blood pressure of SHR（P<0.05）,and the hypotensive degree of PD-M was greater than that of API control group but less than that of positive control group.There was no significant long-term effect on systolic blood pressure,diastolic blood pressure,mean blood pressure,heart rate and body weight of SHR in blank micelle group（P>0.05）.Conclusion:Preparation of PUE into PD-M by membrane hydration method can improve the solubility,oral bioavailability and antihypertensive effect of PUE on SHR rats. |
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