| Puerarin（Pue）is also called puerarin flavone,is a sourced flavonoid glycoside from the leguminous plant Pueraria lobata（Willd.）Ohwi or Pueraria edulis Pamp.Researches have demonstrated that Pue has dramatically biological activities,which are mainly reflected in antioxidation,anti-inflammatory,hypoglycemic,anti-tumor,regulate blood sugar,treatment of liver fibrosis and so on,which makes it attract close attention in medicine and other fields.Nevertheless,Pue has low bioavailability,because of the poor water solubility and membrane permeability（BCS class IV）,short half-life and intestinal first-pass metabolic effect,which restricts the clinical application.Glycyrrhizic acid is a kind of saponins with natural surface activity in licorice.The research aims to prepare puerarin nanoemulsion lyophilized powder（Pue-NE-LP）based on natural stabilizer glycyrrhizic acid to improve the bioavailability of Pue.The research used high-energy emulsification method to prepare puerarin nanoemulsion（Pue-NE）.In order to guarantee the curative effects of the formulation and to suppress the affection of instability mechanisms and environmental factors on the stability of Pue-NE,the Pue-NE-LP was further prepared by freeze-drying technology.In the early stage,the formulation and process parameters of Pue-NE were preliminarily screened through single factor study,and then the majorized formulation was obtained through the central composite design-response surface method,also the physicochemical property,features of external release as well as storage stability of Pue-NE prepared with the best formulation were investigated.Later period,Pue-NE-LP was successfully prepared by screening the type and amount of lyophilization protectant,and the physical and chemical properties,in vitro dissolution and influencing factors of Pue-NE-LP were investigated.The specific research contents are as follows:This project work created the HPLC content analysis method of puerarin,which lays a solid foundation for the preliminary quality evaluation of Pue preparations.The high-energy emulsification method was used to prepare Pue-NE based on the natural stabilizer glycyrrhizic acid.The single-factor method determined the process as follows:high-speed shearing speed 19 000 r/min,high-pressure homogenizing pressure 100 Mpa,homogenizing 6 times.Optimize the formulation with the central composite design-response surface method to determine the dosage of Pue was 0.5 wt%,the dosage of glycyrrhizic acid was 0.175 wt%,and the dosage of oil-phase caprylic acid glyceride was 4.375%（v/v）.The physicochemical properties and stability inspection results reveal that Pue-NE is milky white uniform and flowing liquid;it is identified as O/W emulsion by dyeing method;the particle size measured by the laser particle size analyzer is（184.5±0.8）nm,and the PDI is 0.088±0.002,Zeta potential is（10.56±0.35）m V;the results of transmission electron microscope（TEM）observation showed that there was no adhesion between the milk droplets,and they were spherical in uniform size with good formability;the solubility of Pue in Pue-NE measured by HPLC was（4.970±0.008）mg/m L,and the average drug loading was（99.4±0.2）（n=3）%;the p H value measured by the p H meter method is 6.750±0.005（n=3）;the conductivity of Pue-NE measured by the conductivity method is（98.3±0.4）μs/cm（n=3）;according to the turbidity calculation formula,the turbidity is（24.3±1.0）cm-1（n=3）.The release results showed that the cumulative release rate of Pue in distilled water was 87.5%,which was significantly higher than the 45%of physical mixture,indicating that the preparation of Pue into nanoemulsion could improve its release rate in vitro.In addition,the release effect of Pue-NE was the best in PBS at p H 6.8,with the maximum cumulative release of 92%.The results of the centrifugal stability test showed that after 60 minutes of centrifugation at 4000 r/min,Pue-NE did not undergo aggregation,coagulation,phase separation and demulsification,indicating that the centrifugal stability of the emulsion was good;the results of storage stability tests showed that the various indexes of the emulsion increased at the low temperature of4℃and the high temperature of 60℃,but the various indexes of the emulsion did not change significantly at the room temperature of 25℃,suggesting that Pue-NE should be stored at room temperature.Intense light test results showed that Pue-NE system absorbed the heat generated by light,resulting in slight changes in various indexes,suggesting Pue-NE should be kept away from light.Pue-NE-LP was successfully prepared by freeze-drying technology under the premise of using lyophilized protectant.The curing process was investigated by a single factor,and the lyophilized protectant was glucose with a dosage of 5%.The average particle size,PDI and RDI of Pue-NE-LP were（241.1±2.5）nm,0.136±0.008and 1.294±0.006,respectively,compared with that before loop drying,the particle size and PDI of Pue-NE-LP increased by 29.5%and 47.8%respectively;the mean drug loading of Pue in Pue-NE-LP was（41.8±1.3）mg·g-1（n=3）by HPLC;the results showed that the cumulative dissolution rate of Pue-NE-LP in distilled water was more than 90%,which was significantly higher than that of physical mixture freeze-dried powder,indicating that Pue-NE-LP could improve the dissolution rate of API in vitro;in addition,Pue-NE-LP had the best dissolution effect in PBS at p H6.8,and the maximum cumulative dissolution was 96.8%during the study period;Scanning electron microscope（SEM）observation results demonstrated that Pue-NE-LP was irregular and small flake and the size was relatively uniform;X-ray diffraction（XRD）analysis results show that Pue exists in an amorphous state in Pue-NE-LP;Fourier transform infrared spectroscopy（FT-IR）analysis results show that the original structure of Pue remains in the freeze-dried powder,and the molecular structure of Pue does not change during the whole curing process;the influence factor test results show that Pue-NE-LP is stable in high temperature and strong light environment,but it is slightly hygroscopic in high humidity environment with a relative humidity of75%,indicating that Pue-NE-LP should be stored in a dry place. |
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