【作者】 郝秀华；

【导师】 崔福德； 程刚；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2005， 博士

【摘要】 灯盏乙素（scutellarin），4′，5，6-三羟基黄酮-7-O-β-D-葡萄糖醛酸苷，又名野黄芩苷，为灯盏花中提取的黄酮类成分灯盏花素的主要有效成分。具有扩张微血管、改善微循环、提高心肌功能和心脑供血、降低血粘度、抑制血小板聚集、防栓及溶栓、降低血脂和降血糖等作用，临床上主要用于心脑血管等疾病的治疗。本研究首次对灯盏乙素在大鼠胃肠道内的吸收、代谢机制进行探讨，采用体外孵化及直接检测体内胃肠残留的研究方法确定了灯盏乙素在胃肠道内受到肠内菌代谢，阐明吸收过程中存在肠内循环、肠肝循环。通过离体小肠代谢研究证实灯盏乙素受肠上皮细胞代谢。采用微粒体孵化法确定了灯盏乙素在肝、肠微粒体中存在脱去葡萄糖醛酸和葡萄糖醛酸化两种代谢途径。通过肝门静脉给药与尾静脉给药比较，首次证实了肝首过不是灯盏乙素生物利用度低的重要原因；采用在体小肠分泌实验确定了外排载体的存在对灯盏乙素生物利用度的影响较小；采用外翻肠囊法证实了透膜性较差及小肠部位的广泛代谢是其生物利用度低的重要原因。本文以大鼠为研究对象，对灯盏乙素的体内动力学规律进行初步研究：大鼠静脉给予三个剂量的灯盏乙素溶液剂，血药浓度时间曲线相互平行；药时曲线下面积AUC_0-t与剂量成正比，即在10-40 mg／kg剂量的范围内，灯盏乙素在大鼠体内具有线性动力学特征。然而研究发现，灯盏乙素口服生物利用度很低。分析造成其生物利用度低的可能原因主要有以下几个方面：胃肠液中溶解度较低，膜的渗透性较差，小肠外排载体的影响，肠道吸收前代谢转化，肝首过代谢以及肠壁的代谢转化（Ⅰ相和Ⅱ相反应）等。为了探讨灯盏乙素胃肠道吸收、代谢机制，本文分别从以下几个方面进行较为系统的研究：一、首先对灯盏乙素的理化性质进行研究，以确定药物本身性质对其生物利用度的影响，同时研究了灯盏乙素在不同条件下的稳定性，以确保其生物药剂学与药物动力学研究数据的科学性和可靠性。灯盏乙素随着pH值的升高溶解度增大，同时稳定性降低；其油／水分配系数随pH值的升高而迅速降低，苷元的油／水分配系数明显高于灯盏乙素，因此苷元的脂溶性强于原形药物。二、通过大鼠灌胃给予的灯盏乙素溶液剂和混悬剂，比较灯盏乙素溶解状态对生物利用度的影响。灯盏乙素混悬剂和溶液剂绝对生物利用度分别为4.94％和5.65％，经方差分析药时曲线下面积没有明显的区别，说明药物的溶解与否并不是造成生物利用度低的主要原因。三、首过效应包括胃肠首过和肝首过。有首过效应的药物往往生物利用度很低，因此，本研究通过肝门静脉给药与尾静脉给药比较，考察肝首过对灯盏乙素生物利用度的影响。结果肝首过并不是造成灯盏乙素口服生物利用度低的主要原因。四、具有苷键的药物在胃肠道内易受肠内菌代谢影响，而灯盏乙素为野黄芩素的葡萄糖醛酸苷，含有糖苷键，因此本研究采用体外孵化法考察灯盏乙素在胃肠内容物中的代谢。灯盏乙素在盲结肠内容物中迅速被代谢，主要代谢产物为野黄芩素，体外孵化与胃肠残留结果一致，说明灯盏乙素受肠内菌代谢。但肠内菌代谢并不是生物利用度低的主要原因。大鼠单剂量灌胃给予40 mg／kg剂量的灯盏乙素混悬液后，主要通过粪排出体外，仍有大部分药物被吸收。吸收后的药物在脏器器官分布中以肝、肾分布较高。原形药物灯盏乙素在胃、十二指肠、空肠、回肠分布较多，苷元在回肠、盲肠、结肠、直肠分布较多，与药物在胃肠道内代谢产生苷元的浓度大小有关。五、为了考察转运载体介导的小肠分泌对灯盏乙素吸收的影响，本研究采用了单灌流法，通过静脉给药方式对灯盏乙素在体小肠分泌进行研究，通过胆管引流消除胆汁排泄的影响。实验发现在小肠中的分泌总量不到给药剂量的1％，说明外排载体的存在对灯盏乙素生物利用度影响较小。通过小肠外排到肠腔的药物受到肠壁酶或肠内菌代谢释放出苷元，小肠分泌的药物及其代谢物又可以重新被吸收形成肠内循环。胆汁中药物浓度明显高于血浆药物浓度，说明胆汁排泄存在着主动转运的分泌机制。胆汁排泄的药物及其代谢物在消化道中重新被吸收返回肝门静脉，形成肠肝循环。六、小肠是一个重要的首过器官，药物在小肠不仅受到肠内菌作用产生吸收前首过代谢，在吸收过程中进入肠系膜静脉血之前也能够被代谢，本研究采用了离体小肠外翻肠囊法考察灯盏乙素在大鼠小肠的吸收和代谢。结果表明灯盏乙素渗透系数较低，透膜性较差，是其生物利用度低的一个重要原因。通过LC-MS-MS初步鉴定了受药体系小肠液中的代谢产物：M0确定为灯盏乙素，M2确定为野黄芩素（scutellarein），M1推测可能为野黄芩素-6，7-O-β-D-二葡萄糖醛酸苷（scutellarein-6，7-O-β-D-diglucuronide），M3推测可能为野黄芩素-6-O-β-D-葡萄糖醛酸苷（scutellarein-6-O-β-D-glucuronide）。离体小肠代谢产物与肝门静脉血中鉴定的代谢产物相同。说明药物吸收的同时发生了代谢转化，进一步证实了肠壁的代谢是其生物利用度低的重要原因。七、肝脏是药物代谢的重要器官，是机体进行生物转化的主要场所，哺乳动物小肠是物质吸收的主要位点。Ⅰ相和Ⅱ相代谢酶及一些转运载体在此均有表达。采用肝肠微粒体孵化实验考察灯盏乙素在微粒体中的代谢。体外肝、肠微粒体代谢结果表明：NADPH和NADH两种辅助因子对灯盏乙素的代谢没有影响；孵化体系Ⅰ为微粒体氧化反应体系，孵化体系Ⅱ为微粒体葡萄糖醛酸化反应体系。灯盏乙素在肝、肠微粒体孵化体系Ⅰ中，脱去葡萄糖醛酸，主要代谢产物为苷元；在孵化体系Ⅱ同时发生脱去葡萄糖醛酸和葡萄糖醛酸化两种代谢途径，主要代谢产物为苷元和另一葡萄糖醛酸结合物M3；野黄芩素在孵化体系Ⅱ中主要代谢产物为灯盏乙素和M3，说明灯盏乙素在肠壁的代谢转化对其生物利用度的影响显著。更多还原

【Abstract】 Scutellarin, a flavone glucuronide, as 4’, 5, 6-trihydroyflavone-7-O-β-D-glucuronide, extracted from a Chinese herb Erigero breviscapus(vant.) hand.-Mazz., is of particular interest because of its significant dilating blood vessel, improving microcirculation, increasing cerebral blood flow, decreasing blood viscosity, preventing and dissolving thrombus, inhibiting platelet aggregation activity, reducing blood fat, and having hypoglycemic action and so on. Up to date, it has been widely used in treatment of cardiovascular and cerebrovascular diseases.This study contributes to the understanding of mechanisms responsible for low oral systemic availability of scutellarin for the first time and can help identify potential factors affecting the systemic exposure of scutellarin. As a result, incubation method in vitro was adopted and the detection was carried out in gastrointestinal remnant in vivo, indicating that scutellarin was metabolized by intestinal microflora. Metabolism in isolated intestine was studied, and results showed that scutellarin was metabolized by enterocyte cell. Intra-intestinal cycle and entero-hepatic cycle were elucidated along with drug absorption. Extensive metabolism via deglucuronidation and glucuronidation occurred in rat liver and intestinal microsomes using microsomal preparations. Scutellarin was administered to rats by intravenous or intraportal infusion. The data strongly suggested that hepatic first-pass elimination did not exert a significant role in the low oral bioavailability for the first time. In addition, intestinal secretion mediated by transporter played an insignificant role in the presystemic elimination using an in vivo intestinal perfusion model. Limited membrane permeability and extensive metabolism in gut wall might contribute more significantly to the low oral bioavailability of scutellarin using everted sacs method. In our studies, rats were used as the research animal, and the primary pharmacokinetic characteristics of scutellarin were studied. After intravenous administration to rats over the doses range of 10-40 mg/kg, the AUC values were linear over the administered doses range (r=0.995), suggestive of the linear pharmacokinetic characteristic of scutellarin in rats after intravenous administration. However, the bioavailability of scutellarin in rats after oral administration was very poor. The possible reasons of low oral bioavailability of scutellarin were the factors of gastrointestinal tract such as the low oral solubility in gastrointestinal fluid, limited membrane permeability, intestinal secretion mediated by transporter, gut metabolism before absorption, hepatic first-pass elimination, or gut wall metabolism (Ⅰphase reaction andⅡphase reaction). In order to investigate the absorption mechanism of scutellarin, systemic research was carried out in these studies as follows:Firstly, the physicochemical properties of scutellarin were investigated to confirm the effect of essentiality on the bioavailability. Simultaneously, the stability of scutellarin in different conditions was studied to ensure that the pharmacokinetics data were scientific and reliable. Results showed that the solubility of scutellarin was improved with the pH enhancing, and that apparent octanol/water distribution coefficients (log P) decreased with the pH increasing between 1 and 7.4. The log P of scutellarein was higher than that of scutellarin, indicating that scutellarein exhibited a much greater lipophilicity toward membrane permeation than scutellarin.Secondly, rats were administered with scutellarin solution or suspension to investigate the effect of solubility of scutellarin on the bioavailability. After rats were administered scutellarin solution or suspension. The absolute bioavailability of scutellarin solution and suspension were 4.94%, 5.65%, respectively. Moreover, there is no difference between scutellarin solution and suspension through analysis of variance (ANOVA). Results showed that whether it was soluble or not wasn’t the main reason of low oral bioavailability.Thirdly, first pass effect consists of gastrointestinal first-pass effect and hepatic first-pass effect, and the bioavailability of drugs with first-pass effect usually is very low. In the present study, the systemic exposure of scutellarin following intraportal was compared with intravenous administration to understand the contribution of presystemic hepatic elimination to the low oral bioavailability. Results showed that the hepatic first-pass elimination of scutellarin played an insignificant role in the presystemic elimination of orally administered scutellarin.Fourthly, the intestinal microflora is responsible for the liberation of aglycone from glycosidic drug. Scutellarin is a glucuronide of scutellarein which has glycosidic bond. The metabolism of scutellarin in gastrointestinal content was investigated using incubation method in vitro. Results in vitro were consistent with that in vivo, indicating that scutellarin was destroyed seriously in gastrointestinal track after oral administration, and the main metabolite was scutellarein. In other words, scutellarin was metabolized by caecal and colonic microflora. The contribution of the enterobacterium metabolism to the low oral bioavailability of scutellarin did not play a significant role.Results showed that scutellarin and its metabolites were mainly eliminated through feces excretion after a single dose of scutellarin suspension (40 mg/kg), and a majority of drug was absorbed. The distributions of scutellarin in organs at different time after oral administration of scutellarin (100 mg/kg) were different. The concentrations of native scutellarin in liver and kidney were higher than other organs. The distributions of scutellarin in stomach, duodenum, jejunum and ileum were higher than other tissues, but the distributions of scutellarein in ileum, cecum, colon and rectum were higher than other tissues. Drug distribution was related to the metabolic concentration of scutellarin in gastrointestinal tract.Fifthly, in order to investigate the contribution of intestinal secretion mediated by transporter on the oral bioavailability, intestinal secretion in situ perfusion was studied after intravenous administration with a single-pass perfusion technique. The contribution of scutellarin in bile was excluded by catheterization of the biliary duct before perfusion. The intestinal excretion amount of scutellarin in administered dose was lower than 1%. Results showed that the contributions of intestinal secretion on the oral bioavailability exerted insignificant role. Drugs excreted through intestine can be metabolized to liberate aglycone, and then the aglycone can be reabsorbed in intestine to form intra-intestinal cycle. The concentration of scutellarin in bile was higher than that in plasma, and the active excretion mechanism may be present in bile excretion. Scutellarin and its metabolites excreted by bile, parts of which were reabsorbed in intestine into portal vein and entero-hepatic cycle came into being.Sixthly, because of the intestine being an important first-pass organ, drugs were metabolized by intestinal microflora before absorption, and metabolism occurred in the intestine before entering into mesenteric vein. In our studies, intestinal absorption and metabolism of scutellarin in the isolated rat intestine were studied using everted sacs method. Results showed that the apparent permeability coefficient was lower. Therefore, the epithelial permeability of the drug in vivo is predicted to be very low and potentially bioavailability limiting.The metabolites were identified by LC-MS-MS. Metabolite M0 was scutellarin and M2 was scutellarein, M1 may be Scutellarein-6, 7-O-β-D-diglucuronide, M3 may be Scutellarein-6-O-β-D-glucuronide. The metabolites absorbed in intestinal fluid were same as that in portal vein. Results showed that metabolism occurred during the intestinal absorption, and the contribution of gut wall metabolism played a significant role on the low bioavailability.Lastly, liver, as an important drug-metabolizing organ, is the important site for the biotransformation of drugs. The mammalian small intestine serves principally as the site for absorption of nutrients, water, and both beneficial and potentially harmful xenobiotics. Both phaseⅠand phaseⅡmetabolic enzymes are expressed in the small intestine, together with associated transporters. In our studies, the biotransformation of scutellarin in vitro was investigated in rat liver and intestinal microsomes. Results showed that NADPH and NADH had no effect on the metabolic rate of scutellarin in rat liver and intestinal microsomes. Scutellarin was deglucuronidated in rat liver and intestinal microsomes in incubation systemⅠand the main metabolite was scutellarein (M2). Scutellarin was deglucuronidated and glucuronidated in rat liver and intestinal microsomes in incubation systemⅡ, and the main metabolites were scutellarein (M2) and another glucuronide of scutellarein (M3). Scutellarein was glucuronidated in rat liver and intestinal microsomes in incubation systemⅡand the main metabolites were scutellarin and another glucuronide of scutellarein (M3). Results suggested that the biotransformation of scutellarin in gut wall affected the oral bioavailability of scutellarin, evidently.更多还原