| Objective: To extract and purify high purity ellagic acid and geniposide extract from Fructus Canarii and Fructus Gardeniae. To prepare a novel nano lipid ultrasound microbubbles loaded Ellagic acid and Geniposide, then evaluate the enhancement effects by animal experiment. Methods: To determinat content of ellagic acid and geniposide from Fructus Canarii and Fructus Gardeniae by HPLC. Using Fructus Gardeniae and Fructus Gardeniae as the crude material. To inspect the influence of particle size, extraction solvent, solvent concentration, extraction temperature, extraction time, ratio of material to liquid on ellagic acid and geniposide extract purity by the single factor experiment. To purify the Ellagic acid and Geniposide by High speed counter current chromatography and macroporous resin in order to obtain high purity extract. A certain proportion of ellagic acid extract, geniposide extract, distearoylphosphatidylcholine(DSPC), dipalmitoylphosphatidylethanolamine(DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid(DPPA), glycerol, phosphate buffered saline were mixed in a tube. Perfluoropropane gas was the package. Then the nano lipid microbubble was prepared by mechanical vibration. The morphous, dispersion, homogeneity and concentration were observed by optical microscope and electron microscope. Its base physical properties like particle size, distribution and surface potential were detected laser particle size instrument and surface potential measuring instrument. The encapsulation rate and drug loading were detected by HPLC. At the same time, these methods were used to preliminary stability study. To inject self-made nano lipid ultrasound microbubbles through the rabbit ear margin vein, the ultrasound imaging of rabbit liver and tumor in liver were dynamic observed by the ultrasonographic system by self-control method, which provide reliable basis to the ultrasonic targeting drug system. Results: Using ellagic acid and geniposide as reference substances. To determinat content of ellagic acid and geniposide from Fructus Canarii and Fructus Gardeniae by HPLC. ellagic acid and geniposide showed good linearity in the range of 0.043 ~ 2.160μg and 0.419 ~ 20.934 μg. The RSD of the precision,reproducibility and stability tests were less than 3 %. The average recoveries of ellagic acid was 97.69% and the RSD was 1.17%(n=9). The average recoveries of geniposide was 98.75% and the RSD was 0.81%(n=9). Throughing single factor experiment, the best extraction process of ellagic acid was that Fructus Canarii was crushed to fine powder, then adding material to liquid ratio of 1:12(g:m L) 80% acetone solution, 80℃ reflux extraction 2 hours. Finally, the purity of ellagic acid extract was 16.52%. The best extraction process of ellagic acid was that Fructus Gardeniae was crushed to fine powder, then adding material to liquid ratio of 1:5(g:m L) 70% ethanol solution, 80℃ reflux extraction 3 hours. Finally, the purity of geniposide extract was 35.78%. Using isopropyl alcohol, n-butanol and water =1: 2: 3 as the solvent system, To Purify ellagic acid extract by high speed counter current chromatography, The purity of ellagic acid extract was 93.67%, 1.82 g obtained from 100 g Fructus Canarii, the yield was 77.08%. To Purify geniposide extract by X-5 macroporous resin, The purity of geniposide extract was 91.26%, 11.37 g obtained from 100 g Fructus Gardeniae, the yield was 85.69%. The average diameter of the self-made nano lipid microbubbles was(526.8±96.4)nm, the average surface potential was-(5.3±0.9)m V, the average concentration was(3.86±0.72)×109/m L, The encapsulation rate of ellagic acid and geniposide were 92.65±1.32% and 87.39±1.05% respectively, the drug loading were 23.15±0.97% and 21.85±0.83% respectively. The nano lipid microbubbles could greatly enhance the grey scale imaging after intravenous injection. Conclusions: The self-made nano lipid microbubbles has a good shape and uniform particle size distribution. It can enhance the grey scale imaging greatly and provide a protophase foundation for vascular targeting imaging and treatment research of tumors. |
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