| Alzheimer’s disease (AD) is a commonly neurodegenerative disease related with age, accounting for impaired memory, cognitive function decline, and damage to behavior capacity. Resveratrol (Res), a polyphenol deriving from natural plants, has been found to possess potential benefits to human health. Its strong antioxidant effect of scavenging free radicals provides theoretical support for the prevention and treatment of AD. However, the poor solubility of resveratrol and difficult delivery drug to the brain due to the presence of blood brain barrier (BBB) has been the main bottleneck to restrict its application in clinical.The purpose of this subject is to tailor resveratrol nanosuspensions, then considering properties of gellan gum as an ionic sensitive in situ gel to prepare resveratrol nansosuspensions based in situ gel. After intranasal administration, the gel properties were evaluated about their preparation, pharmacokinetic and pharmaco-dynamic character, with the purpose of effective delivery drug to the brain for improvment the bioavailability of resveratrol. This thesis also provides an interesting probe for the treatment of central nervous system disease.The main contents of this study include the following parts:the preformulation study of resveratrol; the process and formulation screening of resveratrol nanosuspensions; Pharmacokinetic characteristics of resveratrol nanosuspensions and the approach to improving its bioavailability; preparation resveratrol nanosuspen-sions based in the ionic sensitive gel for intranasal application; prevention and therapy effects of resveratrol on AD model mice treated with lateral ventricle injection of Aβ25-35 after intranasal administration were assessed through behavior evaluation of mice in water maze experiment and related biochemical indexes change; the protective effects of resveratrol on PC12 cells induced by Aβ25-35 and the possibly involved mechanism were also discussed.In light of UV spectrum results, resveratrol maximum absorption wavelength is determined and the in vitro determination method by HPLC is established, which meets the requirements. According to the results of preformulation study, solubility of resveratrol in water is about 16 μg·mL-1. Liquid water partition coefficient of logP is about 2.3 between octanol and water. According to its characteristics of low solubility and high permeability, the drug belongs to the type Ⅱ drug in BCS classification system. This indicates that slow dissolution resulted from low solubility may be the main reason for poor absorption of resveratrol.The comparison of two commonly used methods for preparing nanosuspensions, high pressure homogenization method and anti-solvent precipitation method, shows that anti-solvent precipitation method is a preferred method to obtain ideal nanosuspensions. This study adopts three factors and three levels of the BBD response surface experiment, taking the particle size and polydispersity index (PDI) as the response. To screen the best prescription, a mathematical model is established and mathematical statistical analysis is used to probe the effects of drug concentration, the amount of stabilizing of PVPK17 and the concentration of surfactant F188 on the response. The optimized formulation is composed of 29.2 (mg/ml) resveratrol,3.63% PVP K17 and 0.38% Poloxamer 188. The freeze-drying curve was analyzed to determine the lyophilization process of nanosuspensions. After the evaluation of appearance, color and redispersibility of lyophilized products,5% mannitol was selected as cryoprotector. Pharmaceutical properties of resveratrol nanosuspensions were studied from many aspects. Laser scattering particle analyzer was used to determine the particle size, polydispersity index, and zeta potential of resveratrol nanosuspensions. The morphology of the raw material and the lyophilized products were analyzed by SEM, the results showed that particle size was 145 ± 6.56 nm, PDI: 0.279, ξ potential:-8.8± 1.52 mV, respectively. The results of the DSC and X-ray diffraction revealed that the crystal form of resveratrol changed into a metastable type or amorphous state in the process of tailor of nanosuspensions. However, infrared spectroscopy (IR) results exhibited that the structure of resveratrol remained trans spatial configuration, which is of great significance to maintain its activity. In comparison with the raw drug, the analysis of dissolution rate is accelerated and the saturated solubility can be improved about 11 times.Compared with coarse suspension, pharmacokinetic results show that the Cmax、 AUMC0'∞ and AUC0'∞ of the rats after oral administration of resveratrol nanosuspensions increased to 3.2 times,1.4 times and 1.33 times, respectively. If coadministration of piperine and resveratrol, these three parameters increased to 1.73, 1.91 and 1.93 times. These results indicate that the bioavailability improvement of resveratrol induced by coadministration of piperine may be related with the inhibition of the activity of glucuronosyltransferase. Also, the Cmax、AUMC0'∞ ' AUC0'∞ in the rats after coadministration of baicalin solid lipid nanoparticles prepared by coacervation technique went up to 161%,147% and 164% respectively, indicating that baicalin can improve the bioavailability of resveratrol.In the preparation process of resveratrol nanosuspensions based in situ gel, the concentration of gellan gum was chosen as ion sensitive gel forming materials according to critical ionic concentration, expanding index and holding water capacity. The results showed that 0.5% gellan gum meets the requirements, with ion-sensitive characteristics but avoiding inconvenience of preparation and use due to high viscosity. Rheology research results show that the in situ gel is a pseudoplastic fluid due to viscosity showing a clear downward trend as shear rate increases; Taking on the characteristics of Frequency sweep experiments and stress tests showed that the rheological type is the typical Cross model, which has the characteristic of shear thinning, thixotropy and yield stress. Compared with gellan gum solution, texture analysis shows when cationic gel formation occurred, its parameters such as density, thick consistency, cohesion and viscosity shows an increasing trend, consistent with the requirements of the gel. The dissolution experiment in vitro suggested that the release of resveratrol nanosuspensions based in situ gel followed the Higuchi model, which may dominated by the matrix diffusion, combing with corrosion behavior.We chose toad palate cilia model, chick embryo chorioallantoic membrane model and pathology section of nasal mucosa in mice model to comprehensively evaluate the toxicity of resveratrol nanosuspensions based in situ gel. The results showed in situ gel has no obvious stimulation to nasal mucosa and no obvious damage as well; indicating the safety of resveratrol nanosuspensions based in situ gel for nasal administration.In comparison with I.V, pharmacokinetics of nasal administration revealed that in brain was 2.88 times, DTE was 458.2% and DTP was 78.18%, respectively. This suggests that nasal administration is an effective approach for brain target.We discussed the improved prevention and treatment effects of resveratrol on AD model induced by lateral ventricle injection of Aβ25-35 through study and memory evaluation in water maze experiment. Behavior assessment showed that in position navigation, escape latency test, each of experimental animals showed a decreased trend with swim training days increase, indicating that in the training process they had the ability of learning and memory in looking for the platform. Compared with the control group, average latency of model group significantly increased. While compared with the model group, treatment group’s average latency was significantly shorter. Space exploration experiment results show that the times of model group going through target quadrant of the platform is less than that of control group. But the times of treatment group with resveratrol are more compared with the model group. As for the changes of cholinergic neurotransmitters, in AD mice brain acetylcholine (Ach) content decreased; the choline acetyltransferase (ChAT) activity decreased, while the activity of acetylcholinesterase (AchE) with the ability to hydrolysis acetylcholine increased. The administration of resveratrol can decrease the activity of acetylcholine enzymes but increase choline acetyltransferase activity and the levels of acetylcholine. The damage to hippocampus neurons can be observed by HE staining and resveratrol produced a protective effect on damaged neurons. Pharmacodynamic results show that resveratrol can ameliorate the declining ability of learning and memory.Resveratrol nanosuspensions and resveratrol raw drug can improve PC 12 cells survival and exert a protective effect against Aβ25-35 induced apoptosis of nerve cells by means of reduction of stress response by inhibiting ROS and regulation of expression of mitochondria-related protein Bax and Bcl-2.In this article, we successfully explored the preparation of resveratrol nanosuspensions based in ion-sensitive in situ gel. After intranasal administration the drug concentration reaching the brain region can be improved, and consequently enhance the prevention and treatment of AD. Therefore, this research lays a theoretical foundation for further development of intranasal formulations. |
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