| Ginkgo biloba extract(Gb E), as yellowish powder, is isolated from the dried leaves of Ginkgo biloba. Gb E, composed of various chemical components, has been widely used in clinic for the prevention and treatment of cardiovascular and cerebrovascular diseases, and central nervous system disease, such as Alzheimer’s disease, vertigo and tinnitus. These benefits of Gb E are presumed to result from the synergistic effect of two distinct groups of compounds: flavonoid glycosides and terpenoid lactones. However, with regard to the oral administration of marketed Gb E products, there remains a critical challenge in the poor clinical therapeutic efficacy of Gb E. Low lipid and water solubility, first pass metabolism, and poor ability to cross blood-brain barrier(BBB) of flavonoid glycosides could be the reasons. Hence, a novel drug delivery system of proliposomes containing a bile salt had been developed to improve the oral bioavailability and the distribution in brain of Gb E, and was characterized in vivo and in vitro. The main content is as follows:1. The development of an analytical method in vitro for P-Gb EA HPLC method was developed for the determination of three flavonoid glycosides(quercetin, kaempferol and isorhamnetin) and four terpenoid lactones(bilobalide, ginkgolides A, ginkgolides B and ginkgolides C) from P-Gb E, respectively, and then was proved by the methodology validation. Besides, a simple, rapid and reproducible ultracentrifugation method was developed for the evaluation of entrapment efficiency(EE) of the reconstituted Gb E liposomes.2. The study on preparation and formulation optimization of P-Gb EP-Gb E was successfully prepared by a modified ethanol injection combined with spray drying method which had many advantages including simple operation, short time-consuming, low cost and large-scale manufacturing. The effects of the formulation concentration, the amount of egg yolk lecithin(e PC), the amount of sodium deoxycholate(Na DC), the type of carrier and the amount of carrier on particle size, size distribution, EEs of flavonoid glycosides and EEs of terpenoid lactones of P-Gb E were investigated by the single-factor test. The amount of e PC, the amount of Na DC and the amount of mannitol carrier were chosen as the three factors remarkably affected the EEs of flavonoid glycosides which were proved as the most important index for the evaluation of P-Gb E. On the basis of the single-factor test, the formulation of P-Gb E was further optimized using the Box-Behnken design and the response surface methodology. The optimal mass ratio of composition was Gb E: e PC: Na DC: mannitol = 200: 225: 67: 735.3. The study on evaluation of P-Gb EP-Gb E was formulated and characterized in terms of morphology, particle size, surface charge, EE, in vitro release study, in vitro dissolution study, molecular state, storage stability and safety. P-Gb E was yellowish powders with little stench. The FE-SEM results showed that the shape of P-Gb E was nearly spherical. The particle size, PDI and zeta potential of P-Gb E were 211.7 ± 5.4 nm, 0.197 ± 0.012, and-27.7 ± 5.5 m V, respectively. The EEs of flavonoid glycosides and terpenoid lactones from P-Gb E were 91.48% ± 0.77%和 96.18% ± 0.55%, respectively. In vitro release study showed that delayed release of flavonoid glycosides and terpene lactones from the reconstituted liposomes, and the release rates were affected by the p H of release medium. The release curves were consistently and well fitted to the model of Korsmeyer-Peppas, and the release was mainly derived from Kick diffusion. In vitro dissolution study showed that enhanced dissolution of flavonoid glycosides and terpene lactones from proliposomes in intestinal tract. The results of DSC and FTIR indicated that a new phase had formed and some weak interactions between Gb E and excipients had occurred in the preparation process of proliposomes. The result of constant temperature acceleration test showed that the storage stability of P-Gb E was good under the condition of the temperature of 40 °C and the relative humidity of 75%. The result of acute toxicity experiment in mice showed that the safety of P-Gb E was well.4. The development of an UPLC-MS/MS method for P-Gb E in biological samplesA rapid, sensitive and accurate UPLC-MS/MS method was developed for the simultaneous determination of quercetin, kaempferol, isorhamnetin, bilobalide, ginkgolides A, ginkgolides B and ginkgolides C in biological samples including rat plasma, six rat tissues(liver, kidney, spleen, heart, lung and brain) and rat intestinal perfusion fluid. The seven analytes were extracted from different biological samples by liquid-liquid extraction using taxifolin as internal standard. Chromatographic separation was performed on a Waters BEH C18(2.1 mm×50 mm, 1.8 μm) column. Electronic spray ion(ESI) was chosen as the ionization source, and the triple quadrupole mass spectrometer was operated in the positive ionization mode using multiple reaction monitoring(MRM) for data acquisition. These methods were fully validated according to the guidance of FDA and SFDA with respect to specificity, linearity, lower limit of quantitation(LLOQ), precision, accuracy, matrix effect, extraction recovery and stability. These results showed that all validation parameters met the requirements of the guidance criteria.5. The study on pharmackinetic of P-Gb E in ratsThe validated UPLC-MS/MS method was applied to the pharmacokinetic study of P-Gb E. Compared with Gb E, the values of Cmax, Tmax, T1/2, AUC0–∞ and MRT0–∞ for quercetin, kaempferol, isorhmnetin, ginkgolide A, ginkgolide B and ginkgolide C from P-Gb E were significantly higher(p<0.05) except bilobalide. The relative bioavailabilities of quercetin, kaempferol, isorhmnetin, ginkgolide A, ginkgolide B, and ginkgolide C from P-Gb E(AUC0–∞) compared with Gb E were 2.45, 2.11, 2.64, 2.03, 3.33 and 2.94, respectively. These results suggested that proliposomes significantly enhanced the absorption in gastrointestinal tract, prolonged the blood circulation time, and improved the oral bioavailability of Gb E.6. The study on tissue distribution of P-Gb E in ratsThe validated UPLC-MS/MS method was applied to the tissue distribution study of P-Gb E. Without regarding to the kidney tissue, seven components from P-Gb E mainly distributed in the tissue of liver, spleen and lung with abundant reticuloendothelial system. Compared with Gb E, the areas under tissue concentration-time curve of seven components from P-Gb E were significantly higher(p<0.05) in liver, spleen, heart, lung and brain, but were significantly lower in kidney(P<0.001). These results suggested that proliposomes could enhance the distribution of Gb E in critical tissues, such as liver, spleen, heart, lung and brain. The orders of relative uptake efficiency of three flavonoid glycosides and four terpenoid lactones from P-Gb E(AUC) compared with Gb E were brain > lung > spleen > heart > liver > kidney, and brain > lung > heart > spleen > liver > kidney, which indicated that proliposomes could selectively distributed in brain and lung, and proliposomes have the ability to cross BBB.7. The study on intestinal absorption mechanism of P-Gb EThe validated UPLC-MS/MS method was applied to the intestinal absorption mechanism study of P-Gb E using the in situ single-pass intestine perfusion model. The absorption constant(Ka) and apparent permeability coefficient(Papp) of quercetin, kaempferol, isorhmnetin, ginkgolide A, ginkgolide B and ginkgolide C from P-Gb E were significantly higher(p<0.05) than Gb E. The order of Ka of seven components from P-Gb E was duodenum > ileum > jejunum > colon. These results suggested that proliposomes could enhance the absorption of Gb E in intestinal tract, and the main absorption sites of proliposomes were duodenum and ileum.In summary, P-Gb E could significantly improve the oral bioavailability, enhance the tissue targeting, and improve the ability to cross BBB of Gb E. Hence, as a novel oral drug delivery system, proliposomes containing a bile salt offer an effect approach to improve the oral bioavailability and the ability to cross BBB of pooly soluble drug, and the study provides a theoretical basis for clinical application. |
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