| Flavonoids is a kind of natural organic compounds which exists widely in nature, it has a variety of pharmacological activities, such as free radical scavenging, anti-oxidation, anti-inflammatory, anti-viral, anti-tumor, anti-hyperlipidemia and anti-osteoporosis. In addition, flavonoids has also been proved to possess the anti-HIV activity. However, the formulation development and clinical application of active ingredients of flavonoids were limited due to its low solubility and stability, poor absorption and bioavailability. Naringenin is naringin aglycone, a kind of natural flavonoids compounds, therefore, naringenin not only inherited the varied pharmacological activities of flavonoids compounds but also flawed its disadvantages of physical and chemical properties. On the basis of nano drug delivery system, liposome was used as drug carrier in this study, the naringenin loaded liposome was prepared through thin-layer dispersion method to increasing the solubility and improving the oral bioavailability of naringenin. Meanwhile, due to the rapid absorption characteristics of naringenin in vivo, cholesterol was replaced with Ergo-PEG to prepare naringenin long-circulating liposome to prolong the in vivo circulation time of the drug, thus to provide theoretical basis for the preparation study of naringenin. The paper was divided into the following five parts:Chapter I ReviewThis chapter was mainly divided into two parts: the first part was reviewed the research progress on active ingredients of flavonoid in recent years according to the classification of nano-preparation, including liposome, polymer micelle, nanoparticles, microemulsion/self-microemulsion, nanocapsules and nanospheres; the second part was summarized the research progress of naringenin, including its physical and chemical properties, pharmacological activities, preparation research statusand so on, which provide theoretical support for the selection of research topics.Chapter II Preparation and in vitro evaluation of naringenin loaded liposomeThe HPLC method of in vitro analysis of naringenin was established firstly in this part, and meanwhile the equilibrium solubility of naringenin was examined in double distilled water and other different p H media(p H 1.2, p H 4.5, p H 6.8). The results showed that the equilibrium solubility ofnaringenin in double distilled water was only 37.64 ± 0.66 μg·m L-1 which indicated that the naringenin was poorly water soluble drug. Subsequently, the naringenin loaded liposome was prepared by thin-layer dispersion method, and then the formulation of liposome was optimized through the single factor experiment combined with orthogonal experiment which using the drug loading efficiency as reference index and the encapsulation efficiency as main evaluation index. The optimized formulation was as follows: the total amount of phospholipids and cholesterol was 350 mg, the ratio of phospholipids and cholesterol was 6:1, the amount of sodium cholate and IPM was 200 mg and 150 mg respectively, the dosage of naringenin was 20 mg and the hydration medium was 5 m L of p H 7.4 PBS solution. The encapsulation efficiency of obtained naringenin loaded liposome was reached to 72.2%, and the drug loading efficiency was 2.44%. Moreover, the in vitro properties of optimal liposome were also studied, the results showed that the particle size was 70.53 ± 1.3 nm and the polydispersity index was 0.259 ± 0.078, the results of particle size distribution were consisted with the observed particle morphology. The zeta potential was-37.4 ± 3.2 m V with higher absolute value, indicating thatthe stability of naringenin loaded liposome was good which was consistent with the stability experiment. The in vitro release results showed that the liposome could significantly improve the release rate and solubility of naringenin in different media which demonstrated that the naringenin loaded liposome could achieve the purpose of increasing the solubility of naringenin.Chapter III Preparation and in vitro evaluation of naringenin long-circulating liposomeIn this part, cholesterol was placed by Ergo-PEG which was synthesized by ergosterol and PEG1000 to prepared the naringenin long-circulating liposome, in order to prolong the circulation time of drug in vivo and thus to get better drug efficacy. Single factor test and orthogonal experiment method were combined to optimize the naringenin long-circulating liposome, the optimized formulation was as follows: the total amount of phospholipids and Ergo-PEG was 400 mg, the ratio of phospholipids and Ergo-PEG was 1: 1, the amount of sodium cholate and IPM were both 200 mg, the ratio of drugs and phospholipid with Ergo-PEG was 1:20, the hydration medium was 10 m L of p H 7.4 PBS solution. The encapsulation efficiency of obtainednaringenin long-circulating liposome was 95.67%, which has been significantly improved compared to naringenin loaded liposome.In vitro properties of optimal naringenin long-circulating liposome were investigated including particle size, zeta potential, transmission electron microscopy, stability and in vitro drug release characteristics. The results showed that the average particle size was 168.57 ± 3.8 nm, the polydispersity index was 0.187 ± 0.048, which was larger thannaringenin loaded liposome. Zeta potential was-33.5 ± 4.1 m V and the potential absolute value is larger than 15 m V which suggested that naringenin long-circulating liposome was obtained good stability, what is more, this conclusion could also be proved in the results of preliminary stability experiments. The results of in vitro drug release of naringenin long-circulating liposome showed that the cumulative drug release rate about 72 h in three kinds of media were all exceed 90%, indicating that long circulating liposome was more likely to increase the solubility of naringenin. Chapter IV The establishment of in vivo analysis method of naringenin and its preparationIn vivo HPLC method was established to determinate the concentration of naringeninin plasma, the plasma samples were treated with precipitate protein and enzymatic hydrolysis method respectively to determine the free naringenin content(X) in plasma and the total content of naringenin(X ’) in plasma. The analysis was carried out on a HPLC system equipped with a Symmetry C18 analytical column, the mobile phase was 30% acetonitrile-phosphate buffer, the detection wavelength was 288 nm, the column temperature was 30°C, the flow rate was 1.0 m L?min-1 and the injection amount was 20 μL. The results demonstrated that the method enjoyed high accuracy and specify, and other indicators such as the recovery rate and stability of the frozen samples met the requirement in vivo drug analysis.Chapter V The in vivo pharmacokinetic study of naringenin and its preparationThe pharmacokinetic parameters after single-dose gavage administration(90 mg?kg-1) of free naringenin, naringenin loaded liposome and naringenin long-circulating liposome to SD rats were investigated.The Tmax of free naringenin, naringenin loaded liposome and naringenin long-circulating liposome was 0.25 h、0.25 h and 0.25 h(free naringenin content), 0.083 h、0.25 h and 0.25 h(the total content of naringenin) respectively, indicating that naringenin can be rapidly absorbed to achieve maximum plasma concentration.Meanwhile, naringenin loaded liposome and naringenin long-circulating liposome with respect to the relative bioavailability of free naringenin were 688.80%, 1187.88%(free naringenin content) and 1343.99%, 2090.83%(the total content of naringenin), respectively. These results suggesting the bioavailability was significantly increased with these two preparations after orally administering three doses in rats, naringenin long-circulating liposome showed better effect compared with naringenin loaded liposome.Compared with naringenin loaded liposome andnaringenin long-circulating liposome, the MRT value of free drug in vivo increased from 9.49 ± 0.44 h to 14.58 ± 0.99 h; t1/2 was extended from7.51 ± 0.55 h to 13.18 ± 1.02 h; the MRT value of the total content of naringenin in vivo increased from 9.34 ± 0.50 h to 15.35 ± 1.17 h; t1/2 was extended from 7.51 ± 0.55 h to 13.18 ± 1.02 h, and the area under curve of naringenin long-circulating liposome was larger than others which demonstrated that naringenin long-circulating liposome could enhance the bioavailability and prolong the circulation time of naringeninmeanwhile, thus to obtain better drug efficacy. |
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