| As is well known, China is the one of the countries with the longest history of using spices. The spices, with a variety of remarkable pharmacological effects, can be used as food additives as well as medicine in the history of mankind. Due to the abundant spice resources in our country, different spices with unique characteristics play an increasing important role in the field of disease prevention and control. Spices have become a major source of clinical treatment drugs, as supported by the research findings in modern pharmacology, epidemiology and modern pharmacology.Over the past few decades, spices have been extensively investigated in preserving flavor, stability and other fields, however, limited studies have been conducted so far to understand the microencapsulation and efficient biological use of the spice active ingredient. Given the special physiochemical property, many active ingredients of spices were limited in developing clinical drugs caused by its poor solubility, high irritation and low bioavailability. Thus, to solve the bottleneck issue on the highly efficient drug delivery and the microencapsulation technologies of spices, we used capsaicin as a model drug to perform a series of studies on its formulation, evaluation and improvement of bioavailability. Advanced methods in the modern pharmaceutics, food science and modern detection techniques were utilized to study the capsaicin-loaded nanoformulation, long-term carriers and the new encapsulating methods. In addition, the high bioavailability and antioxidant activity in vivo of capsaicin-loaded innovative microencapsulation formulation was evaluated in our study.Chapter1Progress of spice active ingredients and the applications in pharmacyThis section reviewed the research progress related to pharmacologic action and modern microencapsulation formulation of spice active ingredients. A detailed summary of the development status on some relevant modern preparations with special regard to the spice active ingredients contained pharmacological activity was given. In addition, the research status of its nanoformulation, long-term controlled release carriers and the new encapsulating methods based on electrospinning was described in this section. Finally, the topic basis and design of this dissertation were presented, which provided a foundation for the development of our work.Chapter2In vitro detection method, purification process and basic properties of capsaicinIn order to provide a theoretical basis for capsaicin-loaded microencapsulation formulation, this section was aimed at the method for detecting capsaicin in vitro, the optimal extraction, separation and purification process, the solubility as well as the in vitro anti-tumor activity of capsaicin. In this section, the simple and effective HPLC method was established and validated for guaranteeing quality of capsaicin. These basic investigations laid the foundation for the research of capsaicin-loaded microencapsulation formulation.1. The establishment of in vitro detection method for capsaicin. The methodology HPLC was established to determine the contents of capsaicin in samples in vitro. In this study, the in vitro assay was turned to be in good specificity with a linear range of1～200μg·mL-1(n=5, r=0.9998). And the intra-day and inter-day precision coefficients of variation (RSD%) both were less than2%. The average recoveries of the analytes were between98.2%and100.9%.2. Extraction, separation and purification process. Capsaicin of purity more than98%was extracted by diethyl ether and purified by the three-stage purification process which contained macroporous resin, silica gel column and C8column. The resulted capsaicin monomer was white powder, with its molecular formula being C18H27NO3. The ESI-MS was conducted to determine the [M+Na]+and [2M+Na]+which were328and633, respectively, resulting in the molecular weight of capsaicin (305). Additionally, studies involving13C-NMR and’H-NMR were performed to confirm the monomer structure of this compound.3. Determination of the basic properties of capsaicin. The solubility of free capsaicin and standardized substance in four different media (double distilled water, pH1.2HC1solution, pH4.5phosphate buffer solution and pH7.4phosphate buffer solution) were measured respectively by equilibrium solubility method. The results showed that capsaicin is a poorly water soluble compound because of the scarce solubility (less than50μg·mL-1in different pH water system). In particular, the results obtained in homemade capsaicin were similar to that of the standardized substance. MTT assay was used to examine the in vitro inhibition effect of free capsaicin on several tumor cell lines, including gastric adenocarcinoma cell line SGC and glioma cell line U251. As a result, after72h incubation, the IC50of the proliferation inhibition of the free drug on SGC and U251were52.97μg·ml-1and27.54μg·mL-1, respectively.Chapter3Capsaicin-loaded nano-formulation based on lipid nanoparticle drug delivery system:Preparation, evaluation for the highly efficient bioavailability and antioxidant activity in vivoThree different kinds of nano-formulations including microemulsion, liposome and micelle, were prepared and characterized by the following aspects:particle size distribution, zeta potential, encapsulation efficiency, morphological characteristics, stability and drug release in vitro. A HPLC method was developed to simultaneously determine the concentration of capsaicin in plasma and tissue samples. The irritation test of capsaicin-loaded nano-formulations on gastric mucosa was performed in rats. An acute liver injury model in mice induced by CCl4was established to evaluate the antioxidant activity in vivo.1. Preparation of capsaicin-loaded nanoformulation and its evaluation in vitro. In this study, capsaicin-loaded microemulsion was optimized by the phase diagram method; the liposome was formulated by thin film hydration method; and the mixed micelle system consisting of polyvinylpyrrolidone (PVP)/sodium cholate/phospholipid was constructed. In the case of three kinds of capsaicin-loaded nanoformulation (microemulsion, liposome, and micelle), displaying particle size of53.5±1.6nm,52.2±1.3nm,15.8±0.3nm, with the polydispersity index being0.126±0.074,0.105±0.049and0.097±0.037, respectively. The zeta potential of the three nano-formulations was-6.8±0.6mV,-41.5±2.7mV and-37.7±2.67mV, and the average encapsulation efficiency was found to be85.3±1.1%,81.9±2.4%and90.9±1.8%, respectively. The TEM image demonstrated that all of the capsaicin-loaded nano-formulations were well dispersed as individual particles with spherical shape. Meanwhile, the results of storage stability showed that the optimized prescriptions were stable during storage time and no obvious change was observed in aspect of drug content, encapsulation efficiency and particle size. The result of the in vitro drug release properties assessed by dialysis method indicated that the capsaicin released from the capsaicin-loaded nano-formulations was significantly faster than that of the free capsaicin. Notably, the profiles of drug-loaded nano-formulations in pH7.4phosphate buffer solution demonstrated a relatively slow release profile compared with other media (double distilled water and pH1.2HC1solution).2. The studies of in vivo bioavailability and gastric mucosa irritation. In the bioavailability study, no significant differences were observed in Tmax and t1/2of capsaicin-loaded nano-formulations compared with that of free drug after intraperitoneal injection, while the Cmax and AUCo-2h were greatly improved when encapsulated into the nano-formulations. It was obviously shown that AUCo-2h of three nano-formulations (microemulsion, liposome and micelle) increased to147.1%,240.1%and152.1%, respectively. However, the Tmax, t1/2and MRT of capsaicin-loaded nano-formulations after oral administration were extend compared with unformulated capsaicin, therefore by maintaining the effective blood concentration for a long time. Meanwhile, the AUCo-24h of three capsaicin-loaded nano-formulations was significantly improved with the relative oral bioavailability of the nanoformulation being263.8%,334.9%and241.9%, respectively, compared with the free capsaicin. In addition, it was important to investigate the gastric mucosa irritation of capsaicin by oral administration due to its pungent properties. The photomicrographs of different samples showed that no vacuoles and inflammatory cell infiltration was observed in the formulation group in contrast with the result of the free drug group and control group, suggesting that capsaicin-loaded nano-formulations could effectively reduce gastric mucosa irritation. Therefore, these nano-formulations hold the potential to be developed into some feasible carriers for oral drug delivery.3. In vivo tissue distribution and antioxidant activity studies. A study was conducted in mice to investigate the tissue distribution of nano-formulated and unformulated capsaicin, and the results showed that the nanoformulation system could alter its in vivo distribution. As compared with the unformulated capsaicin, capsaicin-loaded nano-formulations could result in a reduced concentration in the kidney and promotion of the passive hepatic and splenic targeting. The relative stable distribution in the brain for microemulsion was observed at predetermined time intervals by the different administration routes (intraperitoneal injection and oral administration). However, the differences of the drug distribution via intraperitoneal injection and oral administration were that the accumulation of capsaicin was significantly decreased in the lung when the capsaicin was entrapped into the microemulsion or micellar system after oral administration. In addition, the capsaicin-loaded liposome could greatly improve the antioxidant activity in the acute liver injury model in mice induced by CCl4in contrast with the unformulated capsaicin. This effect could be observed clearly from the increase in activities of SOD, GSH-Px and T-AOC, and the decrease in MDA activity in plasma and liver.Chapter4Preparation and evaluation of long-term capsaicin-loaded hydroxyapatite nanoparticlesCapsaicin-loaded hydroxyapatite nanoparticles were prepared by dispersion method assisted with ultrasonic. In this section, preparations using different technologies were optimized with SEM to evaluate its morphology. Meanwhile, XRD and FT-IR were performed to verify the formation of hydroxyapatite structure. Further studies were then conducted on in vitro drug release and in vivo pharmacokinetics of the capsaicin-loaded hydroxyapatite nanoparticles prepared by impregnation method and encapsulation method, respectively.1. Preparation and characterization of the capsaicin-loaded hydroxyapatite nanoparticles. In this section, different factors caused by drying process, preparation technology, the type and concentration of co-precipitator, the concentration of precursor and ultrasonic power were assessed by SEM to observe the morphology of the drug-loaded nanoparticles. The capsaicin-loaded hydroxyapatite nanoparticle with the particle size around50nm was ultimately prepared for further research. The FT-IR and XRD results of the nanoparticle showed weak crystalline structure with less impurity peak and sharp diffraction angles of2θ appeared at25.9°and31.8°respectively, suggesting a high purity and small particle size of the prepared nanoparticles. In addition, the results obtained from FT-IR were also in accordance with the structural feature of capsaicin-loaded hydroxyapatite nanoparticle.2. In vitro and in vivo drug release properties and oral bioavailability. The results in vitro indicated that the nano-formulations prepared with two different methods both could delay the release rate in each of different dissolution media. And the nano-formulations prepared by encapsulation method have a greater retardation effect than that of impregnation method which performed similarly to the free drug. Due to the long-term effect of nano-formulation, the in vivo retention time and bioavailability after oral administration were significantly improved. In case of capsaicin-loaded hydroxyapatite nanoparticles from impregnation method, similar release behavior was observed with the free drug till8h, followed by stable subsequent release. Nevertheless, the time to reach peak concentrations of the nanoparticles prepared by encapsulation method was prolonged to24h, and the relative bioavailability was greatly enhanced as compared with the free drug and the capsaicin-loaded nanoparticle prepared by the other method.Chapter5Novel microencapsulation technique for capsaicin based on electrospinning and the preliminary evaluation in vitro and in vivoIn this study, utilizing the electrospinning method, the microencapsulation complex of capsaicin was obtained using polyvinylpyrrolidone (PVP) K30as a polymer, and evaluated its in vitro drug release behavior and in vivo pharmacokinetics in rats after oral administration. The shape and surface characteristics of the capsaicin-PVP electrospinning complex were observed in SEM images. Additionally, the existential state of capsaicin in electrospinning complex was characterized by XRD.1. The optimization of preparation and drug release properties in vitro. According to the observation by SEM, the optimum parameters were determined as follows: The voltage was10KV with a collection distance of10cm and the flow rate was0.8 mL·h-1with a stainless needle being0.9mm. The prepared electrospinning complex appeared as amorphous forms with a characteristic hump, indicating that the capsaicin was no longer presented as a crystalline material in the formulation. The results of in vitro drug release demonstrated that the cumulative drug release rate within72h in pH1.2HC1solution and double distilled water were about89.4%and95.7%, respectively, compared with100%in pH7.4phosphate buffer solution within48h. Moreover, the release profile of prepared formulation showed a significantly higher release rate than that of free capsaicin. Particularly, the cumulative release rate of capsaicin from the electrospinning complex (75.3%,82.5and76.2%in pH1.2, pH7.4and double distilled water) was superior to that of free drug (20%) within12h. These results revealed that capsaicin-PVP electrospinning complex had an obvious advantage over capsaicin-loaded liposomes and micelles.2. Pharmacokinetics study in rats. The capsaicin electrospinning complex showed a notable improvement of Cmax, MRT and AUCo-24h. No significant difference was found in Tmax of both unformulated and formulated capsaicin. Although the extended ti/2was observed, it was far below than that of three preliminary capsaicin-loaded nano-formulations. Similarly, the relative oral bioavailability of the capsaicin-PVP electrospinning complex was enhanced to219.8%as compared with the free capsaicin, which appeared lower than three capsaicin-loaded nano-formulations mentioned above. |
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