灯盏乙素大鼠体内生物药剂学与药物动力学研究
|
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。
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.
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。
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.
引文
1．云南省药物研究所．灯盏细辛化学成分的研究(第一报)．中草药通讯．1976，11：11-14
2．王勇，庸淑洁，王丽娟．灯盏花素抗肾上腺素实验性心律失常的作用．中国药业．2001，10(1)：30
3．刘兴德，陈运贞．灯盏花素对大鼠缺血-再灌注心肌细胞凋亡和bcl-2 表达的影响．贵阳医学院学报．2004，29(2)：102-104
4．徐理纳．灯盏花素和黄芩素甙对小鼠肠系膜微动脉解痉作用的观察与比较．微循环学杂志．1999，8(4)：36
5．雷开键，黄燮南，吴芹，陆远富，余丽梅．灯盏花素抑制去甲肾上腺素而增强高钾缩血管反应．中国药理学通报．2004，20(9)：1034-1038
6．王丽娟，朱坤杰．灯盏花素对小鼠学习记忆能力的影响．齐齐哈尔药学院学报．1998，19(4)：303-304
7．王雪松，阮旭中，刘买利．灯盏花素对缺血再灌注鼠脑损伤的脑保护作用研究．中成药．2004，24(12)：947-950
8．薛峥，王雪松，阮旭中．灯盏花素对大鼠脑缺血后细胞间粘附分子-1及其mRNA表达的影响．中国病理生理杂志．2004，20(12)：2287-2290
9．庞荣清，潘兴华，龙沛然，覃敏，吴亚玲．灯盏花素对兔血管平滑肌细胞增殖的影响．中国动脉硬化杂志．2004，12(4)：395-398
10．马丽，朱邦豪，陈健文，丘钦英，周家国，关永源．灯盏花素对糖尿病大鼠肾脏氧化应激的影响．中国药理学通报．2004a，20(9)：1030-1033
11．徐辉碧，周林珠，杨祥良，刘宏．灯盏花乙素清除活性氧作用的研究．中药材．2002，25(7)：491-493
12. Liu H, Yang XL, Wang Y, Tang XQ, Jiang DY and Xu HB. Protective effects of scutellarin on superoxide-induced oxidative stress in rat cortical synaptosornes. Acta Pharmacol Sin. 2003, 24(11): 1113-1117
13．王兆钺，方尔笠，阮长耿．新灯盏花素对血小板活化反应的抑制作用．中草药．1989，20(2)：23-25
14．王影，杨祥良，刘宏，唐晓荞．灯盏花素抗凝血作用的研究．中药材．2003，26(9)：656-658
15．雷开键，黄燮南，吴芹，谢笑龙，余丽梅，陆远富．灯盏花素对血小板聚集的效应与细胞内游离钙的关系．遵义医学院学报．2004，27(1)：11-13
16．白娟等．灯盏花液对实验性大鼠肝纤维化防治作用的研究．中国中医信息杂志，2000，7(5)：34-35
17. Yang XF, He W, Lu WH, Zeng FD. Effects of scutellarin on liver function after brain ischemia/reperfusion in rats. Acta Pharmacol Sin. 2003, 24(11): 1118-1124
18．All Abdella，甘璐，刘琼，刘宏，徐辉碧．灯盏花素的抗氧化作用及拮抗硒对大鼠肝脏毒性的研究．中国药理学通报．2003，19(1)：113-115
19．王红，刘琼，Ali Abdella，徐辉碧．灯盏乙素拮抗硒对人肝细胞L-02毒性的研究．中草药．2004，35(4)：413-415
20．王玮，吴莹瑶，卢岩，范书铎．野黄芩甙抗炎作用的实验研究．中国医科大学学报．2003，32(6)：503-504
21．阮志鹏，于卫东．灯盏乙素抗炎作用的实验研究．长治医学院学报．2004，18(2)：88-89
22．马丽，朱邦豪，周家国，文艳铃．灯盏花素对糖尿病大鼠视网膜血流动力学变化的影响．中山大学学报：医学科学版．2004，25(6)：554-556，597
23．杨锦南，胡世兴，陈慷，邓新国，张悦，张晓惠．金钠多、灯盏花素对N-甲基-N-亚硝脲诱导SD大鼠视网膜变性的保护作用．眼科研究．2003，21(6)：605-608
24．吴永贵，林辉，钱浩，赵珉，周典，梁维龙，徐星铭，张伯科．灯盏花素对大鼠糖尿病模型肾组织TGF-β1与CTGF表达影响的实验研究．中国药理学通报．2004，20(9)：1050-1054
25．赵红艳，张瑶，李守拙，王曦，范书铎．野黄芩甙对发热家兔下丘脑及脑脊液中环磷腺苷含量的影响．中国医科大学学报．2003，32(2)：115-119
26．赵铁华，邓淑华，高巍杨，鹤松石，艳华．野黄芩苷镇痛作用的实验研究．中国中医药信息杂志．2001，8(6)：28-29
27．付美玉，李晓明．灯盏花素注射液治疗缺血性脑血管病疗效对照观察．云南中医药杂志．1999，22(3)：17
28．周靖求．康圣灯盏花素片治疗脑血栓形成84例．中国中医急症．1996，5(5)：207-208
29．王丽萍，郑修广．灯盏花素治疗急性脑出血的临床疗效观察．中国脑血管病杂志．2004，1(10)：445-447
30．何茶叶，刘平．灯盏花素治疗急性脑梗死30例疗效观察．新乡医学院学报．2001，18(6)：425-426
31．乔香兰，关颖，杨丽波，王海超，盛宝英．灯盏花素治疗急性脑梗死的临床研究．黑龙江医药科学．2001，24(3)：45-46
32．孙武，孙永良，孙莉．灯盏花素治疗心绞痛50例．医药导报．2000，19(3)：229
33．陈莉莉．灯盏花素治疗冠心病的疗效观察．大连大学学报．2001，22(2)：97-99
34．杨永华，朱伟荣，朱建秀．灯盏花素治疗血瘀型冠心病30例临床研究．中西医结合心脑血管病杂志．2003，1(3)：141-142
35．张威．灯盏花注射液治疗心绞痛38例体会．现代中西医结合杂志．2001，10(7)：675
36．王智杰，魏向阳，魏蒙．灯盏花素治疗冠心病心绞痛的疗效观察．现代中医药．2004，(6)：16-17
37．雷闽湘，易红，孙志湘，钟慧菊，吴静．灯盏花素治疗糖尿病周围神经病变的疗效观察．湖南医科大学学报．2002，27(4)：337-340
38．虞炳庆，黄明章，芮晓东，刘保群．灯盏花素治疗2型糖尿病患者周围神经病初步观察．河南实用神经疾病杂志．2004，7(2)：8-9
39．张品雅．灯盏花素片治疗中风后遗症32例临床分析．中成药．1998，20(1)：26-27
40．辛复兵，代斌．云南灯盏花注射液治疗内耳眩晕38例．医药导报，1999，18(5)：340
41．郭宏，刘森平，曾健，王士贞．灯盏细辛注射液与常规西药治疗突发性耳聋随机对照研究．中国中医药信息杂志．2004 11(2)：113-114
42．柳玉瑾，邰杏芳，黄飞华，夏翔．灯盏花素片治疗老年痴呆35例临床观察-附对照组25例．辽宁中医杂志．1997，24(6)：263-264
43．韩艳玲，王青，焦朝艳．灯盏花素注射液对高粘滞血症的疗效观察．实用神经疾病杂志．2004，7(5)：64-65
44．李学信，徐庆友．云南灯盏花注射液降血脂的临床观察．临床荟萃．1994，9(21)：1000
45．李慧，邓瑞锋，王曙光，杨红海，彭玲，刘富深，刘海淑．云南灯盏花治疗老年肾功能不全的疗效观察．辽宁中医杂志．2001，28(5)：293
46．陈洪滔，刘洪萍，刘靖，何永成，万启军．灯盏花注射液对慢性肾功能不全患者肾功能的保护．河南职工医学院学报，2003，15(1)：21-22
47．王开江，盛燚．灯盏花素注射液治疗肺心病68例．临床肺科杂志．2002，7(1)：13
48．谢远见，黄金忠．手法配合灯盏花素治疗椎动脉型颈椎病38例．中国民间疗法．2003，11(10)：28-29
49．陈咏萍．灯盏花素对肝硬化微循环和纤维化指标的影响．黑龙江医药科学．2000，23(5)：10-11
50. Santos-Buelga C, Scalbert A. Proanthocyanidins and tannin-like compounds: nature, occurrence, dietary intake and effects on nutrition and health. J Food Sci Agric. 2000, 80: 1094-1117
51. Scalbert A, Williamson G. Dietary intake and bioavailability of polyphenols. J Nutr. 2000, 130(8S Suppl): 2073S-2085S
52. Harborne JB, Williams CA. Advances in flavonoid research since 1992. Phytochemistry. 2000, 55: 481-504
53. Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet. 1993, 342(8878): 1007-1011
54. Knekt P, Jarvinen R, Reunanen A, Maatela J. Flavonoid intake and coronary mortality in Finland: a cohort study. Br Med J. 1996, 312(7029): 478-481
55. Keli SO, Hertog MG, Feskens EJ, Kromhout D. Dietary flavonoids, antioxidant vitamins, and incidence of stroke: the Zutphen study. Arch Intern Med. 1996, 156(6): 637-642
56. Dorant E, Van den Brandt PA, Goldbohm RA, Sturmans F. Consumption of onions and a reduced risk of stomach carcinoma. Gastroenterology. 1996, 110(1): 12-20
57. Knekt P, Jarvinen R, Seppanen R, Hellovaara M, Teppo L, Pukkala E, Aromaa A. Dietary flavonoids and the risk of lung cancer and other malignant neoplasms. Am J Epidemiol. 1997, 146(3): 223-230
58. Middleton E Jr, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev. 2000, 52(4): 673-751
59. Xu X, Wang HJ, Murphy PA, Cook L, Hendrich S. Daidzein is a more bioavailable soymilk isoflavone than is genistein in adult women. J Nutr. 1994, 124(6): 825-832
60. King RA, Broadbent JL, Head RJ. Absorption and excretion of the soy isoflavone genistein in rats. J Nutr. 1996, 126(1): 176-182
61. King RA, Bursill DB. Plasma and urinary kinetics of the isoflavones daidzein and genistein after a single soy meal in humans. Am J Clin Nutr. 1998, 67(5): 867-872
62. Setchell KD, Brown NM, Desai P, Zimmer-Nechemias L, Wolfe BE, Brashear WT, Kirschner AS, Cassidy A, and Heubi JE. Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J Nutr. 2001, 131 (Suppl 4): 1362-1375
63. Kurzer MS, Xu X. Dietary phytoestrogens. Annu Rev Nutr. 1997, 17: 353-381
64. Baba S, Furuta T, Fujioka M, Goromaru T. Studies on drug metabolism by use of isotopes ⅩⅩⅦ: urinary metabolites of rutin in rats and the role of intestinal microflora in the metabolism of rutin.J Pharm Sci. 1983, 72(10): 1155-1158
65. Bokkenheuser VD, Shackleton CH, Winter J. Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans. Biochem J. 1987, 248(3): 953-956
66. Schoefer L, Mohan R, Schwiertz A, Braune A and Blaut M. Anaerobic degradation of flavonoids Clostridium orbiscindens. Appl Environ Microbiol. 2003, 69(10): 5849-5854
67. Kyoichi K and Teruaki A. Relation of intestinal Bacteria to Pharmacological effects of glycosides. Bioscience Micro flora.1997, 16(1): 1-7
68. Rechner AR, Smith MA, Kuhnle G, Gibson GR, Debnam ES, Srai SK, Moore KP, Rice-Evans CA. Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products. Free Radic Biol Med. 2004, 36(2): 212-225
69. Lee NK, Choi SH, Park SH, Park EK and Kim DH. Antiallergic activity of hesperidin is activated by intestinal microflora. Pharmacology. 2004, 71.(4): 174-180
70. Zhang QY, Dunbar D, Ostrowska A, Zeisloft S, Yang J, Kaminsky LS. Characterization of human small intestinal cytochromes P-450. 1999, 27(7): 804-809
71. Liu Y. and Hu M. absorption and metabolism of flavonoids in the caco-2 cell culture model and a perused rat intestinal model. Drug Metab Dispos. 2002, 30(4): 370-377
72. Day AJ, Canada FJ, Diaz JC, Kroon PA, Mclauchlan R, Faulds CB, Plumb GW, Morgan MR, Williamson G. Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase. FEBS Lett. 2000, 468(2-3): 166-170
73. Day AJ, DuPont MS, Ridley S, Rhodes M, Rhodes MJ, Morgan MR, Williamson G. Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glucosidase activity. FEBS Lett. 1998, 436(1): 71-75
74. Hanke DW, Warden DA, Evans 10, Fannin FF, Diedrich DF. Kinetic advantage for transport into hamster intestine of glucose generated from phlodzin by brush border beta-glucosidase. Biochim Biophys Acta. 1980, 599(2): 652-663
75. Ioku K, Pongpiriyadacha Y, Konishi Y, Takei Y, Nakatani N, Terao J. beta-Glucosidase activity in the rat small intestine toward quercetin monoglucosides. Biosci Biotechnol Biochem. 1998, 62(7): 1428-1431
76. Kaminsky LS, Zhang QY. The small intestine as a xenobiotic-metabolizing organ. Drug Metab Dispos. 2003, 31(12): 1520-1525
77. Rein D, Lotito S, Holt RR, Keen CL, Schmitz HH. and Fraga CG. Epicatechin in human plasma: in vivo determination and effect of chocolateconsumption. J Nutr. 2000, 130(85 Suppl): 2109-2114
78. Hackett AM, Griffiths LA, Broillet A, Wermeille M. The metabolism and excretion of (+)-[14C] cyanidanol-3 in man following oral administration. Xenobiotica. 1983, 13(5): 279-286
79. Richelle M, Tavazzi I, Enslen M, Offord EA. Plasma kinetics in man of epicatechin from black chocolate. Eur J Clin Nutr. 1999, 53(1): 22-26
80. Boutin JA, Thomassin J, Siest G, Cartier A. Heterogeneity of hepatic microsomal UDP-glucuronosyl- transferase activities. Conjugations of phenolic and monoterpenoid aglycones in control and induced rats and guinea pigs. Biochem Pharmacol. 1985, 34(13): 2235-2249
81. Strassburg CP, Nguyen N, Manns MP, Tukey RH. UDP-glucuronosyltransferase activity in human liver and colon. Gastroenterology. 1998. 116(1): 149-160
82. Shali NA, Curtis CG, Powell GM, Roy AB. Sulphation of the flavonoids quercetin and catechin by rat liver. Xenobiotica. 1991, 21(7): 881-893
83. Matsui M, Homma H. Biochemistry and molecular biology of drug-metabolizing sulfotransferase. Int. J. Biochem. 1994, 26(10-11): 1237-1247
84. Floche L. Catechol-O-methyltransferase. Int Pharmacopsychiatry. 1974, 9: 52-60
85. Gee JM, DuPont MS, Day AJ, Plumb GVV, Williamson G, Johnson IT. Intestinal transport of quercetin glycosides in rats involves both deglycosylation and interaction with the hexose transport pathway. J Nutr. 2000, 130(11): 2765-2771
86. Olthof MR, Hollman PC, Vree TB, Katan MB. Bioavailabilities of quercetin-3-glucoside and quercetin-4'-glucoside do not differ in humans.J Nutr. 2000, 130(5): 1200-1203
87. Hollman PC, de Vries JH, van Leeuwen SD, Mengelers MJ, Katan MB. Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Am J Clin Nutr. 1995, 62(6): 1276-1282
88. Hollman PC, van Trijp JM, Buysman MN, van der Gaag MS, Mengelers MJ, de VFies JH, Katan MB. Relative bioavailability of the antioxidant flavonoid quercetin from various foods in man. FEBS Left. 1997, 418(1-2): 152-156
89. Hollman PC, Bijsman MN, van Gameren Y, Cnossen EP, de Vries JH, Katan MB. The sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man. Free Radic Res. 1999, 31(6): 569-573
90. Ader P, Block M, Pietzsch S, Wolffram S. Interaction of quercetin glucosides with the intestinal sodium/glucose co-transporter (SGLT-1). Cancer Lett. 2001, 162(2): 175-180
91. Gee JM, DuPont MS, Rhodes MJ, Johnson IT. Quercetin glucosides interact with the intestinal glucose transport pathway. Free Radic Biol Med. 1998, 25(1): 19-25
92. Crespy V, Morand C, Manach C, Besson C, Demigne C, Remesy C. Part of quercetin absorbed in the small intestine is conjugated and further secreted in the intestinal lumen. Am J Physiol. 1999, 277(1): G120-126
93. Andlauer W, Kolb J, Furst P. Isoflavones from tofu are absorbed and metabolized in the isolated rat small intestine. J Nutr. 2000, 130(12): 3021-3027
94. De Vries MH, Hofman GA, Koster AS, Noordhoek J. Systemic intestinal metabolism of l-naphthol. A study in the isolated vascularly perfused rat small intestine. Drug Metab Dispos. 1989, 17(5): 573-578
95. Koster AS, Noordhoek J. Glucuronidation in isolated perfused rat intestinal segments after mucosal and serosal administration of 1-naphthol. J Pharmacol Exp Ther. 1983, 226(2): 533-538
96. Ayrton A, Morgan P. Role of transport proteins in drug absorption, distribution and excretion. Xenobiotica. 2001, 31(8-9): 469-497
97. Mottino AD, Hoffman T, Jennes L, Cao J, Vore M. Expression of multidrug resistance-associated protein 2 in small intestine from pregnant and postpartum rats. Am J Physiol Gastrointest Liver Physiol. 2001, 280(6): 1261-1273
98. Walgren RA, Walle UK, Walle T. Transport of quercetin and its glucosides across human intestinal epithelial Caco-2 cells. Biochem Pharmacol. 1998, 55(10): 1721-1727
99. Walgren RA, Karnaky KJ Jr, Undenmayer GE, Walle T. Efflux of dietary flavonoid quercetin 4'-beta-glucoside across human intestinal Caco-2 cell rnonolayers by apical multidrug resistance-associated protein-2. J Pharmacol Exp Ther. 2000, 94(3): 830-836
100. Walle UK, French KL, Walgren RA, Walle T. Transport of genistein-7-glucoside by human intestinal CACO-2 cells: potential role for MRP2. Res Commun Mol Pathol Pharmacol. 1999, 103(1): 45-56
101. Vaidyanathan JB, Walle T. Cellular uptake and efflux of the tea flavonoid (-) epicatechin-3-gallate in the human intestinal cell line Caco-2. J Pharmacol Exp Ther. 2003, 307(2): 745-752
102. 葛庆华，周臻，支晓瑾，马丽丽，陈秀华．灯盏花素在犬体内的药动学和绝对生物利用度研究．中国医药工业杂志．2003，34(12): 618-620，632
103. Oliveira EJ, Watson DG. Chromatographic techniques for the determination of putative dietary anticancer compounds in biological fluids. J Chromatogr B 2001 764(1-2): 3-25
104. Zhang JL, Che QM, Li SZ, Zhou TH. Study on metabolism of scutellarin in rats by HPLC-MS and HPLC-NMR. J Asian Nat Prod Res. 2003, 5(4): 249-256
105. Pang KS. Modeling of intestinal drug absorption: roles of transporters and metabolic enzymes (for the gillette review series). Drug Metab Dispos. 2003, 31(12): 1507-1519
106. Osiecka I, Porter PA, Borchardt RT, Fix JA, Cardner CR. In vitro drug absorption models: I. Brush border membrane vesicles, isolated mucosal cells and everted intestinal rings: characterization and salicylate zccumulation. Pharm Res. 1985, 2(6): 284-293
107. Leppert PS and Fix JA. Use of everted intestinal rings for in vitro examination of oral absorption potential. J Pharm Sci. 1994, 83(7): 976-981
108. Kato T, Hayashi Y, Inoue K, Yuasa H. Functional characterization of the carrier-mediated transport system for glycerol in everted sacs of the rat small intestine. Biol Pharm Bull. 2004, 27(11): 1826-1830
109. Polentarutti BI, Peterson AL, Sjoberg AK, Anderberg EK, Utter LM and Ungell AL. Evaluation of viability of excised rat intestinal segments in the Ussing chamber: investigation of morphology, electrical parameters, and permeability characteristics. Pharm Res. 1999, 16(3): 446-454
110．贺云霞，程刚，郝秀华，安峰．尼莫地平大鼠在体肠吸收动力学．沈阳药科大学学报．2004，21(3)：177-180
111. Poelma FG, Tukker JJ. Evaluation of a chronically isolated internal loop in the rat for the study of drug absorption kinetics. J Pharm Sci. 1987, 76(6): 433-436
112. Lennernas H. Human intestinal permeability. J Pharm Sci. 1998, 87(4): 403-410
113. Mottino AD, Hoffman T, Jennes L, Vore M. Expression and localization of multidrug resistant protein mrp2 in rat small intestinel. J Pharmacol Exp Ther. 2000, 293(3): 717-723
114. Nakamura J, Takamura R, Kimura T, Muranishi S and Sezaki H. Enhancement effect of methylxanthines on the intestinal absorption of poorly absorbable dyes from the rat small intestine. Biochem Pharmacol. 1979, 28(19): 2957-2960
115. Gotoh Y, SUZUKI H, KINOSHITA S, HIROHASHI T, KATO Y, SUGIYAMA Y. Involvement of an Organic Anion Transporter (Canalicular Multispedfic Organic Anion Transporter/Multidrug Resistance-Associated Protein 2) in Gastrointestinal Secretion of Glutathione Conjugates in Rats J Pharmacol Exp Ther. 2000, 292(1): 433-439
116. Guo J, Ping Q, Jiang G, Dong J, Qi S, Feng L, U Z, U C. Transport of leuprolide across rat intestine, rabbit intestine and Caco-2 cell monolayer. Int J Pharm. 2004, 278(2): 415-422
117. Lacombe O, Woodley J, Solleux C, Delbos JM, Boursier-Neyret C and Houin G. Localisation of drug permeability along the rat small intestine, using markers of the paracellular, transcellular and some transporter routes. Eur J Pharm Sci. 2004, 23(4-5): 385-391
118. Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemopriotein nature. J of Bio Chem. 1964, 239: 2370-2378.
2．王勇，庸淑洁，王丽娟．灯盏花素抗肾上腺素实验性心律失常的作用．中国药业．2001，10(1)：30
3．刘兴德，陈运贞．灯盏花素对大鼠缺血-再灌注心肌细胞凋亡和bcl-2 表达的影响．贵阳医学院学报．2004，29(2)：102-104
4．徐理纳．灯盏花素和黄芩素甙对小鼠肠系膜微动脉解痉作用的观察与比较．微循环学杂志．1999，8(4)：36
5．雷开键，黄燮南，吴芹，陆远富，余丽梅．灯盏花素抑制去甲肾上腺素而增强高钾缩血管反应．中国药理学通报．2004，20(9)：1034-1038
6．王丽娟，朱坤杰．灯盏花素对小鼠学习记忆能力的影响．齐齐哈尔药学院学报．1998，19(4)：303-304
7．王雪松，阮旭中，刘买利．灯盏花素对缺血再灌注鼠脑损伤的脑保护作用研究．中成药．2004，24(12)：947-950
8．薛峥，王雪松，阮旭中．灯盏花素对大鼠脑缺血后细胞间粘附分子-1及其mRNA表达的影响．中国病理生理杂志．2004，20(12)：2287-2290
9．庞荣清，潘兴华，龙沛然，覃敏，吴亚玲．灯盏花素对兔血管平滑肌细胞增殖的影响．中国动脉硬化杂志．2004，12(4)：395-398
10．马丽，朱邦豪，陈健文，丘钦英，周家国，关永源．灯盏花素对糖尿病大鼠肾脏氧化应激的影响．中国药理学通报．2004a，20(9)：1030-1033
11．徐辉碧，周林珠，杨祥良，刘宏．灯盏花乙素清除活性氧作用的研究．中药材．2002，25(7)：491-493
12. Liu H, Yang XL, Wang Y, Tang XQ, Jiang DY and Xu HB. Protective effects of scutellarin on superoxide-induced oxidative stress in rat cortical synaptosornes. Acta Pharmacol Sin. 2003, 24(11): 1113-1117
13．王兆钺，方尔笠，阮长耿．新灯盏花素对血小板活化反应的抑制作用．中草药．1989，20(2)：23-25
14．王影，杨祥良，刘宏，唐晓荞．灯盏花素抗凝血作用的研究．中药材．2003，26(9)：656-658
15．雷开键，黄燮南，吴芹，谢笑龙，余丽梅，陆远富．灯盏花素对血小板聚集的效应与细胞内游离钙的关系．遵义医学院学报．2004，27(1)：11-13
16．白娟等．灯盏花液对实验性大鼠肝纤维化防治作用的研究．中国中医信息杂志，2000，7(5)：34-35
17. Yang XF, He W, Lu WH, Zeng FD. Effects of scutellarin on liver function after brain ischemia/reperfusion in rats. Acta Pharmacol Sin. 2003, 24(11): 1118-1124
18．All Abdella，甘璐，刘琼，刘宏，徐辉碧．灯盏花素的抗氧化作用及拮抗硒对大鼠肝脏毒性的研究．中国药理学通报．2003，19(1)：113-115
19．王红，刘琼，Ali Abdella，徐辉碧．灯盏乙素拮抗硒对人肝细胞L-02毒性的研究．中草药．2004，35(4)：413-415
20．王玮，吴莹瑶，卢岩，范书铎．野黄芩甙抗炎作用的实验研究．中国医科大学学报．2003，32(6)：503-504
21．阮志鹏，于卫东．灯盏乙素抗炎作用的实验研究．长治医学院学报．2004，18(2)：88-89
22．马丽，朱邦豪，周家国，文艳铃．灯盏花素对糖尿病大鼠视网膜血流动力学变化的影响．中山大学学报：医学科学版．2004，25(6)：554-556，597
23．杨锦南，胡世兴，陈慷，邓新国，张悦，张晓惠．金钠多、灯盏花素对N-甲基-N-亚硝脲诱导SD大鼠视网膜变性的保护作用．眼科研究．2003，21(6)：605-608
24．吴永贵，林辉，钱浩，赵珉，周典，梁维龙，徐星铭，张伯科．灯盏花素对大鼠糖尿病模型肾组织TGF-β1与CTGF表达影响的实验研究．中国药理学通报．2004，20(9)：1050-1054
25．赵红艳，张瑶，李守拙，王曦，范书铎．野黄芩甙对发热家兔下丘脑及脑脊液中环磷腺苷含量的影响．中国医科大学学报．2003，32(2)：115-119
26．赵铁华，邓淑华，高巍杨，鹤松石，艳华．野黄芩苷镇痛作用的实验研究．中国中医药信息杂志．2001，8(6)：28-29
27．付美玉，李晓明．灯盏花素注射液治疗缺血性脑血管病疗效对照观察．云南中医药杂志．1999，22(3)：17
28．周靖求．康圣灯盏花素片治疗脑血栓形成84例．中国中医急症．1996，5(5)：207-208
29．王丽萍，郑修广．灯盏花素治疗急性脑出血的临床疗效观察．中国脑血管病杂志．2004，1(10)：445-447
30．何茶叶，刘平．灯盏花素治疗急性脑梗死30例疗效观察．新乡医学院学报．2001，18(6)：425-426
31．乔香兰，关颖，杨丽波，王海超，盛宝英．灯盏花素治疗急性脑梗死的临床研究．黑龙江医药科学．2001，24(3)：45-46
32．孙武，孙永良，孙莉．灯盏花素治疗心绞痛50例．医药导报．2000，19(3)：229
33．陈莉莉．灯盏花素治疗冠心病的疗效观察．大连大学学报．2001，22(2)：97-99
34．杨永华，朱伟荣，朱建秀．灯盏花素治疗血瘀型冠心病30例临床研究．中西医结合心脑血管病杂志．2003，1(3)：141-142
35．张威．灯盏花注射液治疗心绞痛38例体会．现代中西医结合杂志．2001，10(7)：675
36．王智杰，魏向阳，魏蒙．灯盏花素治疗冠心病心绞痛的疗效观察．现代中医药．2004，(6)：16-17
37．雷闽湘，易红，孙志湘，钟慧菊，吴静．灯盏花素治疗糖尿病周围神经病变的疗效观察．湖南医科大学学报．2002，27(4)：337-340
38．虞炳庆，黄明章，芮晓东，刘保群．灯盏花素治疗2型糖尿病患者周围神经病初步观察．河南实用神经疾病杂志．2004，7(2)：8-9
39．张品雅．灯盏花素片治疗中风后遗症32例临床分析．中成药．1998，20(1)：26-27
40．辛复兵，代斌．云南灯盏花注射液治疗内耳眩晕38例．医药导报，1999，18(5)：340
41．郭宏，刘森平，曾健，王士贞．灯盏细辛注射液与常规西药治疗突发性耳聋随机对照研究．中国中医药信息杂志．2004 11(2)：113-114
42．柳玉瑾，邰杏芳，黄飞华，夏翔．灯盏花素片治疗老年痴呆35例临床观察-附对照组25例．辽宁中医杂志．1997，24(6)：263-264
43．韩艳玲，王青，焦朝艳．灯盏花素注射液对高粘滞血症的疗效观察．实用神经疾病杂志．2004，7(5)：64-65
44．李学信，徐庆友．云南灯盏花注射液降血脂的临床观察．临床荟萃．1994，9(21)：1000
45．李慧，邓瑞锋，王曙光，杨红海，彭玲，刘富深，刘海淑．云南灯盏花治疗老年肾功能不全的疗效观察．辽宁中医杂志．2001，28(5)：293
46．陈洪滔，刘洪萍，刘靖，何永成，万启军．灯盏花注射液对慢性肾功能不全患者肾功能的保护．河南职工医学院学报，2003，15(1)：21-22
47．王开江，盛燚．灯盏花素注射液治疗肺心病68例．临床肺科杂志．2002，7(1)：13
48．谢远见，黄金忠．手法配合灯盏花素治疗椎动脉型颈椎病38例．中国民间疗法．2003，11(10)：28-29
49．陈咏萍．灯盏花素对肝硬化微循环和纤维化指标的影响．黑龙江医药科学．2000，23(5)：10-11
50. Santos-Buelga C, Scalbert A. Proanthocyanidins and tannin-like compounds: nature, occurrence, dietary intake and effects on nutrition and health. J Food Sci Agric. 2000, 80: 1094-1117
51. Scalbert A, Williamson G. Dietary intake and bioavailability of polyphenols. J Nutr. 2000, 130(8S Suppl): 2073S-2085S
52. Harborne JB, Williams CA. Advances in flavonoid research since 1992. Phytochemistry. 2000, 55: 481-504
53. Hertog MG, Feskens EJ, Hollman PC, Katan MB, Kromhout D. Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study. Lancet. 1993, 342(8878): 1007-1011
54. Knekt P, Jarvinen R, Reunanen A, Maatela J. Flavonoid intake and coronary mortality in Finland: a cohort study. Br Med J. 1996, 312(7029): 478-481
55. Keli SO, Hertog MG, Feskens EJ, Kromhout D. Dietary flavonoids, antioxidant vitamins, and incidence of stroke: the Zutphen study. Arch Intern Med. 1996, 156(6): 637-642
56. Dorant E, Van den Brandt PA, Goldbohm RA, Sturmans F. Consumption of onions and a reduced risk of stomach carcinoma. Gastroenterology. 1996, 110(1): 12-20
57. Knekt P, Jarvinen R, Seppanen R, Hellovaara M, Teppo L, Pukkala E, Aromaa A. Dietary flavonoids and the risk of lung cancer and other malignant neoplasms. Am J Epidemiol. 1997, 146(3): 223-230
58. Middleton E Jr, Kandaswami C, Theoharides TC. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev. 2000, 52(4): 673-751
59. Xu X, Wang HJ, Murphy PA, Cook L, Hendrich S. Daidzein is a more bioavailable soymilk isoflavone than is genistein in adult women. J Nutr. 1994, 124(6): 825-832
60. King RA, Broadbent JL, Head RJ. Absorption and excretion of the soy isoflavone genistein in rats. J Nutr. 1996, 126(1): 176-182
61. King RA, Bursill DB. Plasma and urinary kinetics of the isoflavones daidzein and genistein after a single soy meal in humans. Am J Clin Nutr. 1998, 67(5): 867-872
62. Setchell KD, Brown NM, Desai P, Zimmer-Nechemias L, Wolfe BE, Brashear WT, Kirschner AS, Cassidy A, and Heubi JE. Bioavailability of pure isoflavones in healthy humans and analysis of commercial soy isoflavone supplements. J Nutr. 2001, 131 (Suppl 4): 1362-1375
63. Kurzer MS, Xu X. Dietary phytoestrogens. Annu Rev Nutr. 1997, 17: 353-381
64. Baba S, Furuta T, Fujioka M, Goromaru T. Studies on drug metabolism by use of isotopes ⅩⅩⅦ: urinary metabolites of rutin in rats and the role of intestinal microflora in the metabolism of rutin.J Pharm Sci. 1983, 72(10): 1155-1158
65. Bokkenheuser VD, Shackleton CH, Winter J. Hydrolysis of dietary flavonoid glycosides by strains of intestinal Bacteroides from humans. Biochem J. 1987, 248(3): 953-956
66. Schoefer L, Mohan R, Schwiertz A, Braune A and Blaut M. Anaerobic degradation of flavonoids Clostridium orbiscindens. Appl Environ Microbiol. 2003, 69(10): 5849-5854
67. Kyoichi K and Teruaki A. Relation of intestinal Bacteria to Pharmacological effects of glycosides. Bioscience Micro flora.1997, 16(1): 1-7
68. Rechner AR, Smith MA, Kuhnle G, Gibson GR, Debnam ES, Srai SK, Moore KP, Rice-Evans CA. Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products. Free Radic Biol Med. 2004, 36(2): 212-225
69. Lee NK, Choi SH, Park SH, Park EK and Kim DH. Antiallergic activity of hesperidin is activated by intestinal microflora. Pharmacology. 2004, 71.(4): 174-180
70. Zhang QY, Dunbar D, Ostrowska A, Zeisloft S, Yang J, Kaminsky LS. Characterization of human small intestinal cytochromes P-450. 1999, 27(7): 804-809
71. Liu Y. and Hu M. absorption and metabolism of flavonoids in the caco-2 cell culture model and a perused rat intestinal model. Drug Metab Dispos. 2002, 30(4): 370-377
72. Day AJ, Canada FJ, Diaz JC, Kroon PA, Mclauchlan R, Faulds CB, Plumb GW, Morgan MR, Williamson G. Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase. FEBS Lett. 2000, 468(2-3): 166-170
73. Day AJ, DuPont MS, Ridley S, Rhodes M, Rhodes MJ, Morgan MR, Williamson G. Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glucosidase activity. FEBS Lett. 1998, 436(1): 71-75
74. Hanke DW, Warden DA, Evans 10, Fannin FF, Diedrich DF. Kinetic advantage for transport into hamster intestine of glucose generated from phlodzin by brush border beta-glucosidase. Biochim Biophys Acta. 1980, 599(2): 652-663
75. Ioku K, Pongpiriyadacha Y, Konishi Y, Takei Y, Nakatani N, Terao J. beta-Glucosidase activity in the rat small intestine toward quercetin monoglucosides. Biosci Biotechnol Biochem. 1998, 62(7): 1428-1431
76. Kaminsky LS, Zhang QY. The small intestine as a xenobiotic-metabolizing organ. Drug Metab Dispos. 2003, 31(12): 1520-1525
77. Rein D, Lotito S, Holt RR, Keen CL, Schmitz HH. and Fraga CG. Epicatechin in human plasma: in vivo determination and effect of chocolateconsumption. J Nutr. 2000, 130(85 Suppl): 2109-2114
78. Hackett AM, Griffiths LA, Broillet A, Wermeille M. The metabolism and excretion of (+)-[14C] cyanidanol-3 in man following oral administration. Xenobiotica. 1983, 13(5): 279-286
79. Richelle M, Tavazzi I, Enslen M, Offord EA. Plasma kinetics in man of epicatechin from black chocolate. Eur J Clin Nutr. 1999, 53(1): 22-26
80. Boutin JA, Thomassin J, Siest G, Cartier A. Heterogeneity of hepatic microsomal UDP-glucuronosyl- transferase activities. Conjugations of phenolic and monoterpenoid aglycones in control and induced rats and guinea pigs. Biochem Pharmacol. 1985, 34(13): 2235-2249
81. Strassburg CP, Nguyen N, Manns MP, Tukey RH. UDP-glucuronosyltransferase activity in human liver and colon. Gastroenterology. 1998. 116(1): 149-160
82. Shali NA, Curtis CG, Powell GM, Roy AB. Sulphation of the flavonoids quercetin and catechin by rat liver. Xenobiotica. 1991, 21(7): 881-893
83. Matsui M, Homma H. Biochemistry and molecular biology of drug-metabolizing sulfotransferase. Int. J. Biochem. 1994, 26(10-11): 1237-1247
84. Floche L. Catechol-O-methyltransferase. Int Pharmacopsychiatry. 1974, 9: 52-60
85. Gee JM, DuPont MS, Day AJ, Plumb GVV, Williamson G, Johnson IT. Intestinal transport of quercetin glycosides in rats involves both deglycosylation and interaction with the hexose transport pathway. J Nutr. 2000, 130(11): 2765-2771
86. Olthof MR, Hollman PC, Vree TB, Katan MB. Bioavailabilities of quercetin-3-glucoside and quercetin-4'-glucoside do not differ in humans.J Nutr. 2000, 130(5): 1200-1203
87. Hollman PC, de Vries JH, van Leeuwen SD, Mengelers MJ, Katan MB. Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers. Am J Clin Nutr. 1995, 62(6): 1276-1282
88. Hollman PC, van Trijp JM, Buysman MN, van der Gaag MS, Mengelers MJ, de VFies JH, Katan MB. Relative bioavailability of the antioxidant flavonoid quercetin from various foods in man. FEBS Left. 1997, 418(1-2): 152-156
89. Hollman PC, Bijsman MN, van Gameren Y, Cnossen EP, de Vries JH, Katan MB. The sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man. Free Radic Res. 1999, 31(6): 569-573
90. Ader P, Block M, Pietzsch S, Wolffram S. Interaction of quercetin glucosides with the intestinal sodium/glucose co-transporter (SGLT-1). Cancer Lett. 2001, 162(2): 175-180
91. Gee JM, DuPont MS, Rhodes MJ, Johnson IT. Quercetin glucosides interact with the intestinal glucose transport pathway. Free Radic Biol Med. 1998, 25(1): 19-25
92. Crespy V, Morand C, Manach C, Besson C, Demigne C, Remesy C. Part of quercetin absorbed in the small intestine is conjugated and further secreted in the intestinal lumen. Am J Physiol. 1999, 277(1): G120-126
93. Andlauer W, Kolb J, Furst P. Isoflavones from tofu are absorbed and metabolized in the isolated rat small intestine. J Nutr. 2000, 130(12): 3021-3027
94. De Vries MH, Hofman GA, Koster AS, Noordhoek J. Systemic intestinal metabolism of l-naphthol. A study in the isolated vascularly perfused rat small intestine. Drug Metab Dispos. 1989, 17(5): 573-578
95. Koster AS, Noordhoek J. Glucuronidation in isolated perfused rat intestinal segments after mucosal and serosal administration of 1-naphthol. J Pharmacol Exp Ther. 1983, 226(2): 533-538
96. Ayrton A, Morgan P. Role of transport proteins in drug absorption, distribution and excretion. Xenobiotica. 2001, 31(8-9): 469-497
97. Mottino AD, Hoffman T, Jennes L, Cao J, Vore M. Expression of multidrug resistance-associated protein 2 in small intestine from pregnant and postpartum rats. Am J Physiol Gastrointest Liver Physiol. 2001, 280(6): 1261-1273
98. Walgren RA, Walle UK, Walle T. Transport of quercetin and its glucosides across human intestinal epithelial Caco-2 cells. Biochem Pharmacol. 1998, 55(10): 1721-1727
99. Walgren RA, Karnaky KJ Jr, Undenmayer GE, Walle T. Efflux of dietary flavonoid quercetin 4'-beta-glucoside across human intestinal Caco-2 cell rnonolayers by apical multidrug resistance-associated protein-2. J Pharmacol Exp Ther. 2000, 94(3): 830-836
100. Walle UK, French KL, Walgren RA, Walle T. Transport of genistein-7-glucoside by human intestinal CACO-2 cells: potential role for MRP2. Res Commun Mol Pathol Pharmacol. 1999, 103(1): 45-56
101. Vaidyanathan JB, Walle T. Cellular uptake and efflux of the tea flavonoid (-) epicatechin-3-gallate in the human intestinal cell line Caco-2. J Pharmacol Exp Ther. 2003, 307(2): 745-752
102. 葛庆华，周臻，支晓瑾，马丽丽，陈秀华．灯盏花素在犬体内的药动学和绝对生物利用度研究．中国医药工业杂志．2003，34(12): 618-620，632
103. Oliveira EJ, Watson DG. Chromatographic techniques for the determination of putative dietary anticancer compounds in biological fluids. J Chromatogr B 2001 764(1-2): 3-25
104. Zhang JL, Che QM, Li SZ, Zhou TH. Study on metabolism of scutellarin in rats by HPLC-MS and HPLC-NMR. J Asian Nat Prod Res. 2003, 5(4): 249-256
105. Pang KS. Modeling of intestinal drug absorption: roles of transporters and metabolic enzymes (for the gillette review series). Drug Metab Dispos. 2003, 31(12): 1507-1519
106. Osiecka I, Porter PA, Borchardt RT, Fix JA, Cardner CR. In vitro drug absorption models: I. Brush border membrane vesicles, isolated mucosal cells and everted intestinal rings: characterization and salicylate zccumulation. Pharm Res. 1985, 2(6): 284-293
107. Leppert PS and Fix JA. Use of everted intestinal rings for in vitro examination of oral absorption potential. J Pharm Sci. 1994, 83(7): 976-981
108. Kato T, Hayashi Y, Inoue K, Yuasa H. Functional characterization of the carrier-mediated transport system for glycerol in everted sacs of the rat small intestine. Biol Pharm Bull. 2004, 27(11): 1826-1830
109. Polentarutti BI, Peterson AL, Sjoberg AK, Anderberg EK, Utter LM and Ungell AL. Evaluation of viability of excised rat intestinal segments in the Ussing chamber: investigation of morphology, electrical parameters, and permeability characteristics. Pharm Res. 1999, 16(3): 446-454
110．贺云霞，程刚，郝秀华，安峰．尼莫地平大鼠在体肠吸收动力学．沈阳药科大学学报．2004，21(3)：177-180
111. Poelma FG, Tukker JJ. Evaluation of a chronically isolated internal loop in the rat for the study of drug absorption kinetics. J Pharm Sci. 1987, 76(6): 433-436
112. Lennernas H. Human intestinal permeability. J Pharm Sci. 1998, 87(4): 403-410
113. Mottino AD, Hoffman T, Jennes L, Vore M. Expression and localization of multidrug resistant protein mrp2 in rat small intestinel. J Pharmacol Exp Ther. 2000, 293(3): 717-723
114. Nakamura J, Takamura R, Kimura T, Muranishi S and Sezaki H. Enhancement effect of methylxanthines on the intestinal absorption of poorly absorbable dyes from the rat small intestine. Biochem Pharmacol. 1979, 28(19): 2957-2960
115. Gotoh Y, SUZUKI H, KINOSHITA S, HIROHASHI T, KATO Y, SUGIYAMA Y. Involvement of an Organic Anion Transporter (Canalicular Multispedfic Organic Anion Transporter/Multidrug Resistance-Associated Protein 2) in Gastrointestinal Secretion of Glutathione Conjugates in Rats J Pharmacol Exp Ther. 2000, 292(1): 433-439
116. Guo J, Ping Q, Jiang G, Dong J, Qi S, Feng L, U Z, U C. Transport of leuprolide across rat intestine, rabbit intestine and Caco-2 cell monolayer. Int J Pharm. 2004, 278(2): 415-422
117. Lacombe O, Woodley J, Solleux C, Delbos JM, Boursier-Neyret C and Houin G. Localisation of drug permeability along the rat small intestine, using markers of the paracellular, transcellular and some transporter routes. Eur J Pharm Sci. 2004, 23(4-5): 385-391
118. Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemopriotein nature. J of Bio Chem. 1964, 239: 2370-2378.