Preparing20(S)-protopanaxadiol-liquid Crystalline Nanoparticles Containing Piperine And Its Polybasic Characteristic Absorption Research

The efficacy of traditional Chinese medicine oral preparation mainly depends on the absorptions of active components in the gastrointestinal tract. But many active components such as scutellarin, nobiletin, silybin and the forth have low oral bioavailability, which restrict their clinical efficacy. The biopharmaceutical properties such as solubility, membrane permeability and first-pass effect are the main factors that affecting drug absorption. Solubility mainly affects the release rate of drug. In digestive tract, membrane permeability determines the transport velocity in epithelium and specific absorption parts of drugs. First-pass effects include gastrointestinal and liver presystemic metabolism has great influence on absorption.The molecular weight of20(S)-protopanoxadiol (PPD) is460.3. It is the aglycone of protopanoxadiol group with the effect on changing the integrity of cancer cell membrane integrity, inducing cancer cell apoptosis and inhibiting cancer cell metastasis, which includes ginsenoside Rbl, Rb2, Rb3, Rc, Rd, Rg3and Rh2. Studies have shown that among all ginsenosides, PPD has the most potent anticancer activity, which is promising in application. The results show that PPD has low solubility, well membrane permeability and low bioavailability. The suitable preparation is needed to increasing its oral bioavailability. Single index is difficult to evaluate the complicated absorption and metabolism in vivo. Therefore in this study, the equilibrium solubility and apparent oil/water partition coefficient, membrane permeability, absorption window, the bioavailability and metabolism were combined to research the absorption properties and metabolic properties in vivo. All of them were used to provide theoretical and practical foundation for design the PPD preparation.The results showed that PPD was poor water solubility, and the equilibrium solubility in water is only35.24mg/L. Oil-water partition coefficients was46.21(logP=1.66). On Caco-2cell model, the results showed PPD uptake in general, and it also has efflux. In situ intestinal perfusion model, the results showed that the absorption of PPD in the intestine is good, and the effective permeability coefficient were duodenum>jejunum>ileum> colon. The oral bioavailability of PPD was29.39%. It was not well. Metabolic studies showed PPD in vivo presented the extensive metabolism. So the main factors restricted oral bioavailability of PPD was due to the poor solubility and first-pass effect.The biopharmaceutical properties of monomeric compounds in traditional Chinese medicine often exists their restractions. It affects the absorption and bioavailability of drugs in the body. Through interactions between natural products, drugs have the function of coordination, complementary or inhibition. It is better than monomeric compounds by its pleiotropy and multi-targets in many ways. The specific performance is changing the biopharmaceutical properties using the synergistic effect (increase solubility, inhibition of efflux, inhibit metabolism etc.) to improve the bioavailability and absorption in vivo, ther promote its efficacy. Therefore, compared with the current monomer preparation, consequence of these interactions has its unique advantages. The alkaloid piperine is a major componentoj black (Piper nigrum Linn) peppers. Piperine has chemopreventive effects and suppresses lung metastasis-induced melanoma and reduces the invasion and migration of tumor cells, which indicates its potential usefulness in anticancer therapy. In addition, piperine inhibits the cytochrome P450function. Compared to the currently marketed cytochrome P450inhibitors, piperine is safe with little side effect. Thus it is an ideal candidate for inhibiting metabolism and then improving the oral bioavailability of PPD.Colloidal dispersions of bicontinuous cubic phases have been proposed as potential drug carriers, because they are bioadhesive, present a permeation enhancer as the structure-forming lipid, and have the ability to incorporate compounds independent of their solubility. In receni years, it has been paid more and more attention. It is usually made of polar lipids in aqueous environment by adsorption of quantitative water spontaneously formed with special internal structure of the gel phase of cubic structure, with mucosal adhesion strong, thus promoting drug membrane absorption. Cubic phases were formed by a monoolein/poloxamer407/water system which could contain PPD and piperine to increase their solubility and promote the compatibility of the intestinal cells. It improves the bioavailability of PPD from promoting absorption and inhibiting metabolism. Cubic nanoparticles loaded with PPD and piperine were prepared by fragmentation of glyceryl monoolein (GMO)/poloxamer407bulk cubic gel and verified using transmission electron microscopy, small angle X ray diffraction and differential scanning calorimetry. We evaluated the in vitro release of PPD from these nanoparticlesThe amount of free available PPD was about70%at12hours. However, the overall release of PPD from the cubic nanoparticles was less than5%at12hours. Similarly, the release of PPD from PPD-cubosome loaded with piperine was less than5%at12hours. This may because of the high affinity of PPD for the hydrophobic domain of the cubic phase that makes the release of PPD from the nanoparticles difficult. Because the overall release of PPD from the cubic nanoparticles was negligible, we did not perform extensive examination of its release from other formulations. It can infer that mechanism which promoting the absorption is not by increasing the dissolution. In vitro Caco-2cell monolayer, PPD-cubosome and PPD-cubosome loaded with piperine (molar ratio PPD/piperine,1:3) increased the apical to basolateral permeability values of PPD across the Caco-2cell monolayer from53%to64%, respectively. The efflux ratio of PPD-cubosome loaded with piperine was not significantly different from that of the PPD-cubosome. This may be because of fewer metabolic enzymes in the Caco-2cell monolayer than in the human body; thus, piperine may not have a significant impact on the efflux ratio. Combining with in vitro release experiments, it can infer that cubosome promotes absorption is by increasing membrane permeability instead of increasing the dissolution. PPD, PPD cubic crystal and PPD piperine cubic liquid crystal in the rat intestinal perfusion model all showed good permeability coefficient, cubic liquid crystal package PPD with lipid cubic liquid crystalline properties, which improve the affinity to the intestinal cells. Thereby, four different intestinal segments in the duodenum, jejunum, ileum and colon can be promoted the absorption of PPD. Compared with no loading of piperine cubic, the permeability coefficient has no significant difference. It may because intestinal perfusion intestinal bacteria metabolism does not exist in this experiment. Meanwhile piperine and PPD were encapsulated in a cubic were pinocytosised by intestinal absorption so that secreted enzyme showed no inhibitory effect. Both PPD-cubosome and the PPD-cubosome loaded with piperine could increase the bioavailability of PPD. The relative bioavailability of the PPD-cubosome loaded with piperine (AUC0-∞) compared to that of PPD (AUC0-∞) and the PPD-cubosome (AUC0-∞) was2.48and1.47, respectively. Meanwhile, the pharmacokinetic parameters clearance (CL) of PPD-cubosome loaded with piperine is lower than that of PPD and PPD-cubosome. It can infer that piperine could inhibit the metabolism of PPD. In this study, ultra-performance liquid chromatography (UPLC)/quadrupole-time-of-flight mass spectrometry (QTOF-MS) was applied to the rapid analysis of20(S)-protopanaxadiol (PPD) metabolites in rats after oral administration, enabling the structural characterization of23metabolites in plasma, bile, urine, and feces.16of these have not been previously reported. The results also indicated the major metabolic reactions of PPD in vivo. UPLC/TOF-MS analysis of plasma samples showed that PPD was extensively metabolized in the plasma. After oral administration of PPD-cubosome, the peak area of PPD in the plasma was significantly higher than that of the controls. In addition, the metabolites of PPD were significantly increased. Thus, on the basis of these results, we can infer that PPD-cubosome increases absorption as well as metabolism. Interestingly, oral administration of PPD-cubosome loaded with piperine reduced the metabolites of PPD. Thus, PPD-cubosome loaded with piperine inhibits the metabolism of PPD. Therefore, in this article, PPD-cubosome loaded with piperine was prepared and the improving the bioavailability of PPD from promoting absorption and inhibiting metabolism.