【作者】 马燕；

【导师】 陈大为； 苏薇薇；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2006， 博士

【摘要】 柚皮素（4’，5，7—三羟基二氢黄酮，naringenin）是广泛存在于植物界的二氢黄酮类化合物，国内外的研究表明柚皮素具有广泛的药理作用。本实验室前期进行了柚皮素药物代谢动力学的研究，研究过程中主要发现了两个问题：一是柚皮素大鼠灌胃给药后所得的血药浓度-时间曲线出现双峰现象；二是柚皮素口服后的绝对生物利用度较低；因此本论文研究的主要目的是：分析柚皮素血药浓度出现双峰现象的原因；通过制剂学和化学结构改造的方法改善柚皮素的口服生物利用度，并分别通过体外Caco-2细胞模型和动物体内试验对两种方法制备的产物进行筛选和验证；并采用Caco-2细胞模型进一步研究柚皮素的口服吸收机制等。本文分别从给药剂量、肠肝循环、胃肠循环三个方面探讨了柚皮素血药浓度-时间曲线出现双峰的原因，结果表明肠肝循环是引起双峰现象的主要原因，其次是胃肠循环，试验设定的高、中、低剂量的血药浓度时间曲线都出现双峰现象，说明剂量不是引起双峰现象的原因。肠肝循环对药物的作用有较重要的影响，它能够增加药物在体内的滞留时间，保证药物在作用部位（或靶器官）有较高的浓度。前期试验研究结果表明，大鼠灌胃给予柚皮素后的绝对生物利用度以游离柚皮素的形式计为3.8％，以柚皮素葡糖醛酸结合物的形式计为39.8％，说明柚皮素的口服生物利用度不高，药物的口服生物利用度低会引起个体差异大、血药浓度波动大，同时增加生产成本，为了提高柚皮素的生物有效性，更好的发挥药效，本文的研究重点在于如何提高柚皮素的生物利用度。通过对柚皮素基本理化性质的考察发现柚皮素的水溶性、脂溶性都不好，分析其自身的理化性质可能是引起生物利用度较低的原因，所以本论文通过制剂学手段和化学结构改造改善柚皮素的理化性质。一是通过制剂学技术，制备了柚皮素磷脂复合物、PVP K 30固体分散体和β-环糊精包合物，柚皮素PVP K 30固体分散体和β-环糊精包合物均有改善柚皮素水溶性的作用，β-环糊精包合物的增溶效果优于PVP K 30固体分散体，包合物使柚皮素在水中的溶解度由71.33μg／ml提高到了793.33μg／ml；制备的柚皮素磷脂复合物明显改善了柚皮素的脂溶性，使柚皮素在氯仿中的溶解度由几乎不溶提高到了1.52±0.21mg／ml，同时水溶性也有所提高，溶解度达到241.71±11.45μg／ml为原来溶解度的3倍；在此基础上本文重点考察了柚皮素磷脂复合物的制备，通过正交试验得到最佳处方和工艺，即投料比为柚皮素／磷脂=1：2，反应温度为40℃，柚皮素的反应液浓度为10％（mg／ml）；按照最佳处方和工艺制备柚皮素磷脂复合物，通过Caco-2细胞模型试验证明磷脂复合物有促进柚皮素吸收的作用，其表观渗透系数(Apparent permeability coefficients，P_app)为15.1±1.18×10^-6cm·s^-1是柚皮素P_app6.80×10^-6 cm·s^-1的2.22倍，提示磷脂复合物会改善柚皮素的体内吸收，提高其生物有效性。二是通过化学结构改造的方法得到柚皮素的两个前体药物Ⅰ和Ⅱ，两者分别改善了柚皮素的脂溶性（Ⅰ）和水溶性（Ⅱ），利用Caco-2细胞模型评价前体药物的吸收并与柚皮素进行比较。柚皮素前体药物Ⅰ和Ⅱ在吸收过程中部分水解产生原型药物柚皮素，部分以前体药物的形式转运，前体药物Ⅰ的P_app是2.19×10^-6 cm·s^-1仅为柚皮素P_app的三分之一，说明其吸收较慢；前体药物Ⅱ的P_app是9.22×10^-6cm·s^-1，表现出较好的吸收特性，而且由于Ⅱ明显改善了柚皮素的水溶性，可以提供更多的给药途径，具有开发利用的价值。利用Caco-2细胞模型进行药物的初筛节约时间，样品处理简单，无需整体动物实验，无种属差异，实验变异性小，本文利用该模型成功筛选出柚皮素磷脂复合物和前体药物Ⅱ；除此之外，本文还利用该模型对柚皮素的口服吸收机制进行了研究，有助于在新药开发的初期就筛选出有开发价值的活性化合物，而且通过了解药物的吸收特性可以为药物的结构改造、剂型和处方设计等提供理论依据。建立了评价口服药物吸收特性的Caco-2细胞模型，并通过显微镜观察细胞形态学、测定跨上皮细胞膜电阻（Transepithelium electrical resistance，TEER）、碱性磷酸酶活性和标记物普萘洛尔的转运特性等指标，对建立的细胞模型进行评价，结果表明本实验建立的Caco-2细胞模型单层膜的完整性、紧密性、碱性磷酸酶活性和渗透性均符合要求，可以用于研究口服药物的吸收机制。本文利用建立的Caco-2细胞模型主要进行了以下试验：①比较了柚皮苷和柚皮素的吸收速度，两者的P_app分别为2.24×10^-6 cm·s^-1和6.80×10^-6 cm·s^-1，柚皮素的P_app是柚皮苷的3倍，提示柚皮素在体内的吸收要快于柚皮苷，因柚皮苷是柚皮素的糖苷，其极性大，不易透过细胞膜转运，故转运速率相对苷元慢；②主要从药物的浓度、药液的pH值、P-糖蛋白（P-glycoprotein，P-gp）的外排和吸收促进剂等方面研究了柚皮素的口服吸收机制及影响因素，结果表明柚皮素的吸收量与药物浓度呈正比关系，不同浓度的P_app无显著性差异（P＞0.05），符合被动扩散的特点；pH值对柚皮素的吸收无影响，不同pH值的P_app无显著性差异（P＞0.05），说明在试验设定的pH值范围内柚皮素无特定吸收；试验研究表明柚皮素的转运过程中存在外排现象，加入P-gp抑制剂维拉帕米后外排现象仍然存在，提示可能是其他载体介导的外排，如多药耐药蛋白（multidrug resisitance-associated protein，MRP）；PEG 400常作为难溶性药物的吸收促进剂，能增加药物的溶解度和提高药物的跨膜转运，试验结果表明PEG 400可以促进柚皮素的跨膜转运吸收；但是Tween 80对柚皮素的吸收有抑制作用，分析可能因为加入Tween 80后降低了磷脂双分子层的流动性，所以影响了药物的吸收。研究表明肠肝循环是引起柚皮素血药浓度中双峰现象的主要原因；Caco-2细胞模型研究表明柚皮素主要以被动扩散方式通过细胞膜转运吸收，同时存在受体介导的细胞外排现象，外排现象也可能是引起柚皮素血药浓度双峰现象和生物利用度较低的原因；制备所得的柚皮素磷脂复合物的体内、外试验均表明其有效的提高了柚皮素的口服生物利用度；通过结构改造得到的前体药物Ⅱ口服吸收后能够较快水解成原型药物柚皮素，而且前药Ⅱ明显改善了柚皮素的水溶性，具有进一步研究开发的潜力。更多还原

【Abstract】 Naringenin, 4’, 5, 7-trihydroxyflavanone, are flavonoids widely existed in the plant kingdom. Previous reports have shown naringenin has many biological and pharmacological activities in home and aboard. The pharmacokinetics studies of naringenin in our lab generated two problems. One was that there existed double peaks phenomenon in the plasma concentration-time curve. The other was the relative low absolute bioavailability after oral administration. So the aim of the article was to analysis how the double peaks phenomenon appeared and to improve the oral bioavailability of naringenin through pharmaceutical methods and chemical structure modification. Moreover, the application of Caco-2 cell model was to study the absorption mechanism of naringenin and offer some basis and new routes for drug structure modification and dosage form design.We further discussed and analysis the double peaks phenomenon from dosages, enterohepatic circulation and gastroenteric circulation. The final result was that the double peaks were mainly due to enterohepatic circulation, then gastroenteric circulation, and dosages had no effects.Previous studies claimed the low oral bioavailability of naringenin. The absolute bioavailability following oral ingestion in rats was 3.8% and 39.8% calculated by free and total naringenin, respectively. The low bioavailability might result in individual differences and increased the production costs. As a result how to improve bioavailability of naringenin is the key to this paper.Naringenin is poorly soluble in water and oil obtained from the investigation of its physical and chemical properties and this may be one of the reasons leading the low bioavailability. So we decided to improve its property by pharmaceutical methods and structure modification.Firstly, the naringenin phytosome, solid dispersion and inclusion compound was prepared and proved to better the water solubility of naringenin. Especially inclusion compound increased the water solubility from 71.33μg/ml to 793.33μg/ml and phytosome obviously improved the solubility in CHCl₃ to 1.52±0.21mg/ml as well as the solubility in water 3-fold than before. On the base we obtained the optimized formulation through orthogonal design. The ratio of naringenin to phospholipids was 1 to 2, reaction temperature was 40℃and the concentration of naringenin was 10％（g/ml）. In Caco-2 cell experiment phytosome prepared by optimized formulation could better the absorption of naringenin and the P_app was 2.22-fold than before, indicating phytosomes might improve the bioavailability of naringenin.Secondly, two naringenin prodrugsⅠandⅡwere obtained by structure modification. They improved the solubility in oil and water, respectively. To investigate the absorption of prodrugsⅠandⅡwith Caco-2 cell model results were that the two naringenin prodrugs could hydrolysis to naringenin during the course of absorption, but the transport of prodrugⅠwas slow due to its high lipophilicity. ProdrugⅡwas well absorbed and well soluble in water with a P_app of 9.22×10^-6 cm·s^-1, so prodrugⅡcan provide more administration routes and have certain exploitation and development potential.By comparison the properties of the obtained products and the Caco-2 cell experiments, it indicated phytosomes and prodrugⅡmight improve the bioavailability of naringenin. The bioavailability experiment of naringenin phytosome in rats was carried out and the research demonstrated phytosome was clearly improved the bioavailability of naringenin. The relative bioavailability of naringenin phytosome was 236％and 184％calculated by free and total naringenin in plasma, respectively. The result was confirmed to Caco-2 cell model. We discussed the in vivo behavior of prodrugⅡin plasma following intravenous administration in rats. ProdrugⅡcould rapidly hydrolysis to naringenin in plasma. Experiment in vivo and in vitro proved prodrugⅡcan be changed into naringenin during the absorption course and have development potential..The Caco-2 cell model was firstly established and evaluated by morphology feature, TEER, alkaline phosphatase and the transport of Propranolol. The determined value was corresponding to the references. And the integrality, permeability and the expression of alkaline phosphatase in the cell model were good. The established Caco-2 cell model can be used to study the absorption mechanism of oral administration drug.The experiments conducted by Caco-2 cell model were as follows:①To compare the absorption rate of naringin and naringenin. The P_app of naringenin was 6.80×10^-6 cm·s^-1, which was 3 fold than that of naringin 2.24×10^-6 cm·s^-1. ②The absorption mechanism of naringenin was studied from concentration, pH value, efflux of P-gp and absorb-promoting agents. There were no obvious differences in P_app both of different concentration and pH. The results showed that the absorption of naringnein was mainly through passive diffusion with possibly carrier-mediated efflux. PEG 400 had the ability to enhance naringenin absorb while Tween 80 inhibited it.The paper systematically studied that enterohepatic circulation was the main reason leading to the double peaks. What’s more, the transport of naringenin was mainly by passive diffusion with efflux which might result in the double peaks and low bioavailability. The obtained phytosomes obviously improved the oral bioavailability of naringenin. While the prodrugⅡinvestigated by experiments in vivo and in vivo was qualified with good behavior following oral administration and having further research and development potential.更多还原