助眠天然活性成分丁香酸脂质体的制备及体内外评价研究

发布时间：2018-05-28 14:45

本文选题：天然药物 + 镇静催眠　；参考：《江苏大学》2017年硕士论文

【摘要】：睡眠是人体重要的生理功能,高质量而充足的睡眠可以有效恢复人体机能。睡眠由中枢神经系统发生并受之调控,同时也受机体与外界多种因素影响。随着现代社会压力的不断增加,失眠现象普遍发生,失眠发病率逐年提高,严重影响着人们的工作生活。西医药物治疗主要为镇静催眠药,存在较大的副作用,如安全性低、依赖性、成瘾性和戒断性等,因而其应用受到限制。近年来研究发现,许多天然药物(中药组方、中药提取物、中药活性成分及天然活性单体)具有良好的镇静催眠作用,而且副作用小,具有良好的开发研究价值。因此,本课题从传统中药中探寻具有改善睡眠作用的活性物质,并对活性成分的纳米制剂进行研究。第一章综述本章对天然药物治疗失眠的研究现状进行综述,重点总结分析了近10年来天然来源的镇静催眠单体及其作用机制的研究进展,展望了镇静催眠中药单体的研究新方向。随着天然产物研究的不断深入,从天然产物中寻找天然助眠活性成分将成为今后失眠研究的重要方向。第二章助眠天然药物JD-XM1提取工艺及活性部位药效研究本章以药效跟踪法,从天然药物JD-XM1中筛选镇静催眠活性部位,并对活性部位药效进行评价。单因素法筛选JD-XM1的提取工艺,优选提取工艺为:乙醇浓度70%、提取温度90 oC、提取时间2 h、液固比12:1。JD-XM1提取物依次用石油醚、二氯甲烷、乙酸乙酯及正丁醇萃取,以阈剂量戊巴比妥钠诱导小鼠睡眠试验评价各极性部位药效,与空白组相比,正丁醇组部位能够显著缩短睡眠潜伏期(182.7±7.6 vs 227.7±29.7 s,P0.01),延长睡眠时间(5518.7±1558.3 vs 2265.8±606.0 s,P0.01)。小鼠自主活动试验和PCPA致小鼠失眠模型评价JD-XM1正丁醇部位药效,结果表明,正丁醇部位能够显著减少活动距离(P0.01)和活动时间(P0.05),且呈现剂量相关性。正丁醇部位可恢复PCPA导致的小鼠失眠行为,与模型组相比,正丁醇部位(300 mg/kg)可缩短戊巴比妥钠诱导的睡眠潜伏期,延长睡眠时间(P0.01)。第三章JD-XM1助眠活性成分的筛选及其结构分析研究本章采用活性跟踪法,从JD-XM1的正丁醇活性部位中,系统分离纯化具有镇静催眠作用的活性单体,并进行结构解析。采用硅胶层析柱对JD-XM1正丁醇活性部位进行分离纯化,得到多种流分。阈剂量戊巴比妥钠诱导小鼠睡眠试验筛选,发现Fr 6流分(二氯甲烷:甲醇=2:1)能够缩短戊巴比妥钠诱导的睡眠潜伏期,延长睡眠时间。C18柱对Fr 6继续纯化,得到白色粉末Compound A,通过NMR和MS进行结构鉴定,为3,5-二甲氧基-4-羟基苯甲酸(丁香酸)。镇静催眠药效实验显示,高剂量丁香酸(200 mg/kg)能够显著延长戊巴比妥钠诱导的睡眠时间(P0.01),缩短睡眠潜伏期(P0.05),并呈现剂量相关性。第四章丁香酸脂质体的制备及其体外评价针对本文筛选出来的JD-XM1活性成分丁香酸,本章建立了HPLC丁香酸的体外分析方法,并进行了方法学考察。结果表明,建立的体外分析方法,系统适用性好,制剂辅料对分析无干扰;在0.5-100μg/m L范围内线性关系良好;48 h内稳定性良好;方法日内/日间精密度高(RSD2%);方法回收率高,可用于丁香酸的定量分析。平衡溶解度实验表明,丁香酸在各种介质中的溶解度均较低,在p H 7.4磷酸缓冲液中的溶解度最大,但仅为78.82±4.68μg/m L,而在水和p H 1.2盐酸溶液中的溶解度接近,分别为66.05±2.65μg/m L和66.86±2.19μg/m L,表明丁香酸的溶解度较差。此外,油水分配系数测定表明,丁香酸log P为1.31,表明丁香酸口服后不易在胃肠道内吸收。以薄膜分散法制备丁香酸脂质体,单因素法筛选处方,优选处方为丁香酸100 mg,大豆磷脂1200 mg,胆固醇200 mg,胆酸钠800 mg,肉豆蔻酸异丙酯800 mg,水化介质为20 m L的PBS(p H 7.4),制得的丁香酸脂质体澄清,包封率高(82.20±2.30%)。体外表征表明,制备的丁香酸脂质体呈类球形,分布均匀,平均粒径为154.67±7.09nm,粒径分布较窄,分散性系数(PDI)为0.14±0.04,Zeta电位为-27.61±3.65 m V。稳定性试验表明,丁香酸脂质体15天稳定性良好,未出现明显的絮凝或浑浊现象,且包封率未有明显变化。第五章丁香酸脂质体大鼠体内药动学及小鼠组织分布研究本章以HPLC建立了测定大鼠血浆及小鼠组织中丁香酸含量的体内分析方法,并进行了方法学考察。在不同生物样品中,线性均良好(r20.99),日内、日间精密度均小于5%,样品回收率良好,符合体内药物检测的方法学要求。丁香酸脂质体的大鼠药动学研究表明,丁香酸脂质体可以提高口服生物利用度。与丁香酸原料药相比,丁香酸脂质体提高了Cmax,给药95 min之后的血药浓度均高于原料药(1.81±0.94 vs 0.12±0.03μg/m L),表明脂质体能够增加药物吸收,且延缓了丁香酸在体内的消除;t1/2显著延长(116.67±14.40 vs 25.36±1.99 min),平均滞留时间显著延长(286.27±18.32 vs 37.98±1.02 min),说明脂质体能够延长丁香酸在体循环时间;丁香酸脂质体显著提高了口服生物利用度,药时曲线下面积AUC0-360min显著提高,为原料药的237.30%。丁香酸脂质体的小鼠组织分布研究表明,丁香酸原料药在血浆中的浓度随着时间的延长快速下降,而脂质体在0.5 h和2 h的血药浓度均显著高于原料药,说明脂质体能够减缓药物消除过程,延长药物在体时间;丁香酸及丁香酸脂质体在各组织器官中药物浓度大小依次为:肾肝肺脾心脑,均趋向于向肾、肝分布;脑部能够检测到丁香酸,说明丁香酸能够透过血脑屏障(BBB),脂质体可减缓药物在脑部的消除,延长药物在脑部的滞留时间,以发挥镇静催眠作用。
[Abstract]:Sleep is an important physiological function of the human body. High quality and sufficient sleep can effectively restore the function of the human body. Sleep is controlled by the central nervous system and is regulated by the body and a variety of external factors. With the increasing pressure of modern society, insomnia is widespread, the incidence of insomnia is increasing year by year, which seriously affects the incidence of insomnia. People's working life. Western medicine is mainly composed of sedative and hypnotic drugs, which have large side effects, such as low safety, dependence, addiction and abstinence, so their application is limited. In recent years, many natural drugs (traditional Chinese medicine, traditional Chinese medicine extract, active ingredients of Chinese medicine and natural active monomer) have good town. The static hypnotic effect, and the side effect is small, has the good development research value. Therefore, this topic seeks to improve the sleep activity from the traditional Chinese medicine, and studies the nanoscale preparation of the active ingredient. The first chapter summarizes the review of this chapter on the research status of insomnia in the treatment of natural drugs, focusing on the analysis of nearly 10 The research progress of the sedative hypnotic monomers and their mechanisms of action of natural sources in the past year, the new direction of the study of sedative hypnotic Chinese medicine monomers is prospected. With the development of natural products, the search for natural hypnotic active components from natural products will become an important direction for the study of insomnia in the future. The second part of the JD-XM1 extraction of natural drugs The study on the efficacy of the active site in this chapter is to screen the sedative hypnotic active parts from the natural drug JD-XM1 and evaluate the active sites. The single factor method is used to select the extraction process of JD-XM1. The optimum extraction process is as follows: the concentration of ethanol is 70%, the extraction temperature is 90 oC, the extraction time is 2 h, and the liquid and solid ratio 12:1.JD-XM1 extract is used in turn. Petroleum ether, dichloromethane, ethyl acetate and n-butanol were extracted with the threshold dose pentobarbital sodium induced mouse sleep test to evaluate the efficacy of each polar part. Compared with the blank group, the n-butanol group could significantly shorten the sleep latency (182.7 + 7.6 vs 227.7 + 29.7 s, P0.01), and prolong the sleep time (5518.7 + 1558.3 vs 2265.8 + 606 s, P0.01). The mouse autonomic activity test and PCPA induced insomnia model evaluated the efficacy of JD-XM1 butanol. The results showed that the location of n-butanol could significantly reduce the activity distance (P0.01) and activity time (P0.05), and showed a dose correlation. The location of n-butanol could restore the behavior of insomnia caused by PCPA, compared with the model group (300 mg/kg). It can shorten the sleep latency induced by pentobarbital sodium and prolong the sleep time (P0.01). Third chapter JD-XM1 the screening and structural analysis of the active components of the hypnotic activity. This chapter uses the active tracking method to separate and purify the active monomers with the sedative and hypnotic effect from the active part of n-butanol in JD-XM1, and analyze the structure. The active parts of JD-XM1 butanol were isolated and purified by the chromatography column. The threshold dose pentobarbital sodium induced a mouse sleep test. It was found that the Fr 6 fraction (dichloromethane: methanol =2:1) could shorten the sleep latency induced by pentobarbital sodium and prolong the sleep time.C18 column to purify Fr 6 and obtain white powder Compound A, Structural identification by NMR and MS for 3,5- two methoxy -4- hydroxybenzoic acid (Ding Xiangsuan). The sedative hypnotic efficacy experiments showed that high dose of syringic acid (200 mg/kg) could significantly prolong the sleep time (P0.01) induced by pentobarbital sodium (P0.01), shorten the sleep latency (P0.05), and present a dose correlation. The preparation of fourth chapter syringic acid liposomes and the preparation of fourth chapters of syringic acid liposome In vitro evaluation of the JD-XM1 active component syringic acid selected in this article, this chapter established an in vitro analysis method of HPLC syringic acid and conducted a methodological investigation. The results showed that the established method of in vitro analysis was good, the preparation excipients did not interfere with the analysis, and the linear relationship in the range of 0.5-100 mu g/m L was good; 48 h was stable. Good sex, high precision (RSD2%) in day / day, high recovery rate and can be used for quantitative analysis of syringic acid. The equilibrium solubility experiment shows that the solubility of syringic acid in various medium is lower and the solubility in P H 7.4 phosphate buffer is the most, but it is only 78.82 + 4.68 Mu g/m L, and the solution in water and P H 1.2 hydrochloric acid solution is dissolved. The degree approach is 66.05 + 2.65 g/m L and 66.86 + 2.19 mu g/m L respectively, indicating that the solubility of syringic acid is poor. In addition, the determination of oil and water distribution coefficient indicates that syringic acid log P is 1.31, indicating that syringic acid is not easily absorbed in the gastrointestinal tract after oral administration. The preparation of syringic acid liposomes by thin film dispersion method is selected by single factor method and the optimum prescription is syringic acid 10 0 mg, soybean phospholipid 1200 mg, cholesterol 200 mg, sodium cholate 800 mg, isopropyl myrisate 800 mg, the hydration medium is 20 m L PBS (P H 7.4), the prepared syringic acid liposomes are clarified and the encapsulation efficiency is high (82.20 + 2.30%). In vitro characterization shows that the prepared syringic acid liposomes are spherical and evenly distributed, the average particle size is 154.67 + 7.09nm, particle size distribution The dispersion coefficient (PDI) was 0.14 + 0.04 and the Zeta potential was -27.61 + 3.65 m V. stability test. The stability of the liposome of syringic acid was good for 15 days, no obvious flocculation or turbidity, and the encapsulation efficiency was not obviously changed. The study on the pharmacokinetics and the tissue distribution of mice in the fifth chapter of syringic acid liposomes was established by HPLC In vivo analysis of the content of syringic acid in rat plasma and mice tissues, and a methodological investigation. In different biological samples, the r20.99 was in good linearity. In the day, the precision of the day was less than 5%, the recovery rate of the sample was good, which accords with the prescription of the drug test in the body. The kinetic study of the rodenticide of syringic acid liposome The syringic acid liposome could improve the oral bioavailability. Compared with the eugenic acid, the syringic acid liposome increased Cmax. The concentration of the blood after 95 min was higher than that of the drug (1.81 + 0.94 vs 0.12 + 0.03 mu g/m L), indicating that the liposomes could increase the absorption of the drug and delayed the elimination of the syringic acid in the body, and the t1/2 was significantly prolonged (1 The average retention time of 16.67 + 14.40 vs 25.36 + 1.99 min) was significantly prolonged (286.27 + 18.32 vs 37.98 + 1.02 min), indicating that liposomes could prolong the body circulation time of Ding Xiangsuan, and the syringic acid liposomes significantly increased the oral bioavailability, and the area AUC0-360min under the curve of the drug was significantly increased, as the 237.30%. syringic acid liposome of the raw material. The study of tissue distribution in mice showed that the concentration of syringic acid in plasma decreased rapidly with time, and the plasma concentration of liposomes in 0.5 h and 2 h was significantly higher than that of the drug. It indicated that liposomes could slow down the process of drug elimination and prolong the time of drug in body; the drug of butylate and syringic acid liposomes were drugs in various tissues and organs. The concentration size is: kidney, liver, lung, spleen, heart and brain, all tend to kidney, liver distribution, the brain can detect Ding Xiangsuan, indicating that syringic acid can pass through the blood brain barrier (BBB), liposome can slow down the elimination of drugs in the brain, prolong the time of drug retention in the brain, and play a sedative and hypnotic effect.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R283.6;R285

【参考文献】