| Piperine is one of the common active ingredient of spices, mainly used as food ingredients and additives in brewing and food industry. Piperine is associated with multiple pharmacological activities such as analgesic, anti-inflammatory, anti-convulsants, anti-tumor and antioxidant, however, given the property, piperine is poorly water soluble and low oral bioavailability which subject a great obstacle to its application in the clinical field. Therefore, micelle and liposome were used as preparation methods respectively in this paper for an enhancement of solubility, stability and oral bioavailability of the piperine, thereby improving the therapeutic index and reducing the side effects of drug. Moreover, these two solubilization methods were compared with each other to provide new strategies for widely application of piperine in future. Herein, the main five parts of the paper were presented as follows:Chapter I ReviewThe review was composed of two parts in this chapter. One was focused on the research advances of solubilizing techniques and their applications to active ingredients of spices, including inclusion technique, solid dispersion technique, emulsion technique, liposome transfection technique and nanoparticle drug delivery system which provided basics for further development and utilization of the active ingredients of spices. Furthermore, the other part were summarized the physical and chemical properties, pharmacological effects of the active ingredient of spices, piperine. Additionally, these two parts of the review were consistent with the keynote of the dissertation which provided theoretical basis for the following experimental studies.Chapter II Preformulation studiesThe analysis method to determine the contents of piperine in samples in vitro was established by HPLC and the methodology validation was carried on meanwhile. In this study, the in vitro assay was turned to be in good specificity with a liner range of 0.5- 100 μg/mL(n=3,R2=0.9997), the limit of quantification(LOQ) and the limit of detection(LOD) of piperine was 14 ng and 5 ng respectively, and the relative standard deviation(RSD) of the method precision was below 2% for inter-day and intra-day calculations. The equilibrium solubility were determined in three different kinds of dissolution media, and the results showed that the equilibrium solubility of piperine was minimum in double distilled water which was only 20.21 μg/m L, while slightly higher in pH 1.2HClsolution and pH 7.4 phosphate buffer solution(only 36.12 μg/m L and 33.69 μg/m L, respectively). Based on these data, it can be concluded that piperine was poorly water soluble which could offer the reference for the further optimization. Chapter III Preparation and in vitro evaluation of PIP-loaded micelleIn this chapter, a phospholipid-sodium cholate-polyvinylpyrrolidone mixed micelle was firstly prepared through thin-film dispersion method, then single factor test and orthogonal design were applied to optimize the prescription and the obtained optimal formulation was confirmed as follows: the dosage of piperine was 5 mg, PIP : phospholipids was 1:80, the amount of cholate and PVP-K30 was both 0.4 g. Secondly, the in vitro properties of PIP-loaded micelle prepared by the optimal formulation were evaluated and the results exhibited that the PIP-loaded micelle was well dispersed as individual particles with spherical shape, the average particle size was about 13.08 ± 0.58 nm, the zeta potential was-22.33 ± 2.73 m V and the encapsulation efficiency was 89.81 ± 1.53%. What is more, the result of preliminary stability test showed that the PIP-loaded micelle had a good stability within 30 days when placed below 60 °C. Furthermore, the in vitro drug release of piperine was test in different dissolution media, it can be found that the release rates of PIP-loaded micelle were all exceeded 95% in 72 h which were significantly higher than free drug, while the cumulative release rate of free piperine was only about 60%, thus the results indicated that micelle could played a solubilization role in piperine.Chapter IV Preparation and in vitro evaluation of PIP-loadedliposomeThe liposome agent were prepared by thin-film hydration method which composed of phospholipid and cholesterol as main carrier, sodium cholate and isopropyl myristate(IPM) added meanwhile. The formulation was optimized by single factor experiment and orthogonal design, and the optimal formulation was obtained as follows: drug : lipid was 1:30, phospholipid: cholesterol was 6:1, the amount of IPM was 0.5g and the amount of sodium cholate was 0.4g. The in vitro properties were evaluated according to the optimal formulation, the results exhibited that PIP-loaded liposome were mostly spherical with a mean particle size of 86.85 ± 1.4 nm, and the zeta potential was-41.4 ± 3.1mV, the encapsulation efficiency was reached to 91.53 ± 0.89%.Additionally, the PIP-loaded liposome observed by the preliminary stability test showed a good stability within 30 days when placed below 60 °C. More important, the in vitro release rate of drug in PIP-loaded liposome was highest(94.03%) in double distilled water in 72 h which increased remarkably by compared with free PIP, therefore it can be demonstrated that the solubility of piperine was significant improved.Chapter V The in situ intestinal absorption characteristics and thepharmacokinetic study of PIP-loaded nanoformulationsThe analysis method to determine the contents of piperine in samples in vivo was established by HPLC based on the SD rats as animal model and the methodology validation was carried on meanwhile. In this study, the in vivo assay was turned to be in good specificity with a liner range of 50- 2500 μg/mL(n=6,R2=0.9971), the relative standard deviation(RSD) of the method precision was below 4% for inter-day and intra-day calculations, the limit of quantification and the limit of detection of piperine was 18 ng and 7 ng respectively, and the average recoveries of the analytes were between 98.77%- 101.21%, RSD was less than 3%. The in situ intestinal absorption characteristics and the pharmacokinetic parameters were evaluated with the conditions of that the PIP-loaded micelle and liposome were regard as test preparation, while free piperine was as reference preparation, the results showed that both PIP-loaded micelle and liposome can enhance the intestinal absorption and oral bioavailability of piperine significantly. On the one hand, the in situ intestinal absorption characteristics illustrated that piperine had absorption in duodenum, jejunum and ileum, but absorption amount and Ka in ileum were significantly higher than that in duodenum and jejunum. Additionally, the largest intestinal drug absorption percentage was only 5.621% when the drug concentration was 10 μg/m L in ileum, indicating that the absorption of free piperine was very poor in intestinal; the constant rate Ka of piperine in ileum were(0.0531 ± 0.059)/h,(0.0598 ± 0.055)/h and(0.0407 ± 0.026)/h respectively, and all the values had significant differences, thus it may be speculated that the passive diffusion and active transport were both existed in the absorption of piperine in small intestine. Furthermore, it can be found that the PIP-loaded micelle and liposome could enhance the in situ intestinal absorption of piperine obviously. On the other hand, the pharmacokinetic study in SD rats indicated that the Cmax of the PIP-loaded micelle and liposome(1368.93ng/m L and 2588.99 ng/mL) in 48 h was improved approximately 1.45-fold and 2.74-fold respectively compared with the free piperine(946.40 ng/m L); the AUC0-48 h of the PIP-loaded micelle and liposome(20336.41 and 51473.85 h·ng/m L) was enhanced 1.44-fold and 3.64-fold respectively; the t1/2 and MRT was prolonged significantly, compared with free piperine, the t1/2 of PIP-loaded micelle and liposome was improved 1.17- 1.60 fold and the MRT was 1.06- 1.34 fold. In conclusion, these results affirmed that the both two kinds of nanoformulation of piperine had the ability to enhance the oral bioavailability of the drug while liposome was much preferably which consistent with the result of in situ intestinal absorption characteristics. Moreover, the IVIVC studies for piperine and its nanoformulation demonstrated good linear relationships between in vitro dissolution and in vivo absorption in three different kinds of dissolution media. |
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