【作者】 周洪伟；

【导师】 王玉蓉；

【作者基本信息】 北京中医药大学 ， 中药制药学， 2012， 博士

【摘要】 本论文主要包括以下几部分：第一部分文献研究对脂质体给药系统的研究进展、生物物理技术用于生物膜相态的研究进展、Caco-2细胞系体外模型及其药学应用的研究进展以及人参皂苷Rgl药理作用与药代动力学的研究进展进行了综述。第二部分实验研究一、人参皂苷Rgl含量测定方法的建立与理化参数研究实验选用Agilent eclipse XDB-C18色谱柱(4.6×250mm,5μm)；乙腈-水(20：80)为流动相；流速0.8mL·min-1;检测波长203nm,柱温25℃；进样体积10μL,结果表明,HPLC法测定人参皂苷Rgl脂质体的包封率,辅料对人参皂苷Rgl的检测没有干扰；以峰面积(Y)对进样量(X)做线性回归,得人参皂苷Rgl回归方程为：Y=455220X-4275.7(R=1),结果表明人参皂苷Rgl在7.415ng-2996.172ng范围内的线性关系良好；人参皂苷Rgl日内RSD为0.35%(n=5)；日间RSD为2.99%(n=5),精密度满足要求；人参皂苷Rgl/DPPC/CH脂质体高、中、低浓度的回收率分别为98.74±3.00%、95.72±3.20%和103.78±2.16%,RSD分别为3.04%、3.34%、2.08%(n=3)；人参皂苷Rgl/DPPC/ST脂质体高、中、低浓度的回收率分别为96.19±0.95%、103.79±3.69%和100.74±5.75%,RSD分别为0.99%、3.56%、5.70%(n=3),回收率良好；考察人参皂苷Rgl、人参皂苷Rgl/DPPC/CH月(?)质体以及人参皂苷Rgl/DPPC/ST脂质体低、中、高浓度在细胞裂解液中的稳定性,结果表明人参皂苷Rgl室温放置24h的RSD分别为0.57%、1.22%和0.86%(n=5)；人参皂苷Rgl/DPPC/CH脂质体室温放置24h的RSD分别为3.18%、2.66%和4.25%(n=5)；人参皂苷Rgl/DPPC/ST脂质体室温放置24h的RSD分别为0.65%、3.88%和0.81%,(n=5),稳定性良好；人参皂苷Rgl药物纯度为99.15%,RSD=0.62%(n=3),药物纯度满足实验要求。通过MarvinSketch 5.9.3软件对人参皂苷Rgl的理化参数进行预测研究,结果经计算得人参皂苷Rgl分子量为800.492207012；人参皂苷Rgl及其电离形式在各pH条件下所占比例结果提示其在体内主要以原型形式存在；logP值为0.68,推测其水溶性稍差；分子中含有约10个H供体与14个H受体,预测人参皂苷Rgl口服吸收较差。二、人参皂苷Rgl脂质体的制备工艺研究考察人参皂苷Rg1(Rg1)(?)旨质体的制备工艺,以包封率为指标优化制备工艺。以二棕榈酰磷脂酰胆碱DPPC).胆固醇(CH)、豆固醇(ST)为膜材,采用薄膜分散法制备Rgl/DPPC/CH脂质体和Rgl/DPPC/ST脂质体。首先进行了单因素考察,对磷脂与固醇比例、药物百分含量、水化时间以及水化温度进行研究,然后通过星点设计-效应面法优化处方工艺,HPLC法测定脂质体中Rgl的包封率,并对脂质体的稳定性进行了考察,结果发现Rg1/DPPC/CH脂质体的制备工艺参数为：药物百分含量为7.4-8.1%,水化时间为50.0-85.0min,水化温度为60.0-63.0℃；Rgl/DPPC/ST脂质体的制备工艺参数为：药物百分含量为7.6-8.1%,水化时间为95.0-100.0min,水化温度为40.0-63.0℃。采用星点设计-效应面法优化Rg1脂质体制备工艺,所建立的数学模型预测性良好,Rgl/DPPC/CH脂质体与Rgl/DPPC/ST脂质体包封率相近,初步提示可用豆固醇替代胆固醇作为人参皂苷Rgl脂质体制备的膜材。三、人参皂苷Rgl及其不同固醇脂质体的分子间相互作用规律研究研究人参皂苷Rgl脂质体中人参皂苷Rgl、磷脂和胆固醇(或豆固醇)三者分子间作用规律。按计量比称取人参皂苷Rgl.DPPC和固醇(DPPC和固醇的摩尔比为2：1),将DPPC和固醇溶于氯仿中,挥尽氯仿后,加入含人参皂苷Rgl的超过量的缓冲溶液(50 mmol·L-1 Tris-HCl,150 mmol·L-1NaCl, 0.1 mmol·L-1 CaCl2,pH 7.2),使水分子与磷脂分子之比为100：1,12000×g离心10min,室温下超声5min,然后将样品在室温和60℃之间进行加热-降温循环处理,最后置4℃冰箱保存24h,得到人参皂苷Rgl脂质体,采用示差扫描量热(DSC)、同步辐射X光衍射(XRD)和拉曼光谱法(Raman)等3种方法,研究脂质体中人参皂苷Rgl、胆固醇(或豆固醇)和二棕榈酰磷脂酰胆碱(DPPC)三者分子间相互作用规律。DSC测定结果表明,随着人参皂苷Rgl浓度的加大,胆固醇脂质体出现分相现象,豆固醇脂质体相态可能发生变化；XRD结果表明,含药量10%的胆固醇脂质体同样出现两个相变信号,豆固醇脂质体变化不明显；拉曼光谱结果表明,随着人参皂苷Rgl的加入,脂质体膜流动性与脂质侧链排列有序性均发生变化。豆固醇脂质体与胆固醇脂质体的相变规律相近,因而进一步证实可用豆固醇替代胆固醇作为人参皂苷Rgl脂质体的膜材。四、人参皂苷Rgl及其脂质体在Caco-2细胞转运研究取冻存的Cago-2细胞,37℃水浴,快速解冻,移至10%FBS的MEM完全培养基,在37℃、相对湿度90%、CO2浓度5%的培养箱内培养,隔日更换培养基,当细胞达到约80%融合时(Caco-2细胞基本铺满瓶壁),用0.25%胰蛋白酶-EDTA消化,按2.0×105/cm2密度将Caco-2细胞接种到12孔Millicell板上。采用跨膜电阻值和Lucifer Yellow CH跨膜转运实验对Caco-2细胞单层模型进行评价；MTT法评价药物对Caco-2细胞的抑制作用,选择溶剂的细胞抑制率<5%,受试物的细胞抑制率<10%,作为合适剂量进行实验；对Caco-2细胞摄取实验的影响因素进行研究,具体包括培养时间对Caco-2摄取的影响、pH对Caco-2摄取药物的影响、温度对Caco-2摄取药物的影响以及药物浓度对Caco-2细胞摄取的影响等,并进行药物跨膜转运实验,结果发现,由于脂质体能够破坏细胞膜中脂质双分子层的有序排列,因而有助于促进药物的吸收,胆固醇脂质体与豆固醇脂质体促进人参皂苷Rgl肠吸收作用相近,该结果再次说明可用豆固醇替代胆固醇作为人参皂苷Rgl脂质体的膜材。五、人参皂苷Rgl及其脂质体的生物利用度评价选取SD雄性大鼠,分别灌服人参皂苷Rgl及其DPPC/CH和DPPC/ST脂质体,于给药后10min、25min、40min、75min、120min、180min、240min、360min和600min眼眶取血,HPLC测定血清中药物含量,计算药物在大鼠体内的药代动力学参数,结果发现,人参皂苷Rgl的两种脂质体Tmax均增长,AUC结果显示人参皂苷Rg1/DPPC/ST(?)旨质体可显著提高原药物在大鼠体内的生物利用度,本研究达到预期目的。更多还原

【Abstract】 The paper includes the following sections:Part 1 Literature ReviewThe paper first of all has summarized the research progress of liposome delivery systems, biophysical techniques for the progress of the biological membrane phase state, Caco-2 cell lines in vitro model and its pharmaceutical applications of research progress, as well as the pharmacological effects of ginsenoside Rgl and its pharmacokineticsthe studies were reviewed.Part 21 Establishment of the determination method for ginsenoside Rgl Determined ginsenoside Rgl by HPLC. The linear range of ginsenoside Rgl was 7.415ng-2996.172ng respectively, and the repeatability,the recovery and stability of the methods met the requirements. MarvinSketch software was selected to predict the physicochemical parameters of ginsenoside Rgl.The molecular weight of ginsenoside Rgl was calculated to be 800.492207012, logP value was 0.68,10 H bond donor and 14 H bond acceptor was contained. All these parameters predicted that ginsenoside Rgl had poor oral absorption.2 Optimization preparation of ginsenoside Rgl liposome by central composite design and response surface methodTo study the preparation method of ginsenoside Rgl (Rgl) liposome and to screen the optimal technological conditions by the encapsulation efficiencies for ginsenoside Rgl,liposomes were made of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (CH) or stigmasterol (ST) by film evaporation technique, then response surface methodology was adopted to screen the optimal conditions. The optimal technological conditions of Rgl/DPPC/CH liposome were as follows:the quality percentage content of Rgl was 7.4～8.1%, the hydration time was 50.0～85.0min,the hydration temperature was 60.0～63.0℃.The optimal technological conditions of Rgl/DPPC/ST liposome were as follows:The quality percentage content of Rgl was 7.6～8.1%, the hydration time was 95.0～100.Omin,and the hydration temperature was 40.0～63.0℃. The central composite design and response surface methodology is suitable for optimizing the formulation. Stigmasterol could be used in the formulation of ginsenoside Rgl liposome.3 Study of interaction between Ginsenoside Rg1 and liposomes employing DSC,XRD and Ramam techniquesTo investigate molecular interaction among ginsenoside Rgl, cholesterol/ stigmasterol and phospholipid in ginsenoside Rgl-encapsulated liposomes, liposomes were made of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol or stigmasterol,and ginsenoside Rgl,then differential scanning calorimetry (DSC),synchrotron x-ray diffraction (XRD) techniques and raman spectra (Raman) were employed to investigate molecular interaction among ginsenoside Rgl, cholesterol/stigmasterol and DPPC in ginsenoside Rgl-encapsulated liposomes. DSC curve displayed two endothermal peaks and curves of SAXS displayed two signals of phase transition when the concentration of ginsenoside Rgl was increased to 20% in ginsenoside Rgl/cholesterol/DPPC liposomes, but in ginsenoside Rgl/stigmasterol/DPPC liposomes,curves of DSC and SAXS just show one signal, Raman spectroscopy results showed that the liposome membrane fluidity changed with the addition of ginsenoside Rg1.Stigmasterol could be used in the formulation of ginsenoside Rgl liposome.4 Transport characteristics of ginsenoside Rgl and its liposome in Caco-2 cell monolayersTaken out the freezing of Caco-2 cells, put them into water(37℃), quickly thawed, then moved to the 10% FBS MEM medium at 37℃,90% of relative humidity and the 5% CO2 concentration. Replaced the medium every other day, when the cells reached approximately 80% confluence (Caco-2 cells basically covered sidewall), digested them with 0.25% trypsin-EDTA.2.0×105/cm2 density of Caco-2 cells were vaccinated to 12-well Millicell board. Transmembrane resistance values and Lucifer Yellow CH transmembrane transport experiments used Caco-2 cell monolayer model to evaluate; MTT method was used to evaluate the drug on the inhibition of Caco-2 cells, selected the solvent inhibition rates<5%, the test substance the inhibition rate of<10%, as the appropriate dose experiments; studied the influencing factors on Caco-2 cell uptake experiments, including the effects of incubation time on uptake in Caco-2 and pH on the intake of drugs in Caco-2, the temperature Caco-2 uptake of drugs and drug concentration on the uptake by Caco-2 cells the impact, and the drug transmembrane transport experiments.The results showed that liposomes can destroy the ordered arrangement of the cell membrane lipid bilayer, thus helping to promote drug absorption, cholesterol liposome and beans steroid liposome for ginsenoside Rgl intestinal absorption was similar.5 Bioavailability Assessment of ginsenoside Rgl liposomesTo assess the Bioavailability of ginsenoside Rgl liposomes, plasma concentrations were determined by HPLC, Kinetica was used to process main pharmacokinetics parameters. The results showed that two kinds of ginsenoside Rgl liposomes’Tmax increased. The AUC results indicated that the ginsenoside Rgl/DPPC/ST liposomes can significantly improve the bioavailability in rats, preparations of ginsenoside Rgl liposomes meets the desired objectives.更多还原