导 师：梅其炳 教授
辅导教师 ：周四元 副教授
中 文 摘 要
方法 （1）采用0.5% CMC-Na将选定的多酚类化合物（芹菜素、大黄酚、大黄素、白藜芦醇）制成50 µmol/kg的混悬液，大鼠灌胃给药后0.08、0.17、0.5、1、2、4、8、12、16、24、36h用乙醚麻醉大鼠，腹静脉采集血样（肝素抗凝），3000rpm离心10min，分离血浆。通过高效液相色谱-质谱连用（LC/MS/MS）方法，采用电喷雾电离负离子模式( ESI- )，多反应监测(MRM)方式检测灌胃给药后，大鼠血浆中多酚类药物浓度随时间变化的规律，同时通过一级全扫描质谱分析和二级质谱分析（子离子扫描），鉴定血浆中多酚类化合物的代谢产物，并通过β-葡萄糖醛酸酶和硫酸酯酶水解的方法，定量分析原形药物、葡萄糖醛酸化和硫酸酯化代谢产物的浓度。Cmax采用实测值，采用梯形法计算AUC值，结合药代动力学软件3P97对所得血药浓度-时间数据进行处理，计算并分析多酚类化合物在大鼠体内代谢的动力学参数。（2）腹腔注射4%的苯巴比妥钠（40mg/kg）麻醉大鼠，尾静脉注射肝素（90 U/ kg）全身抗凝，选取7-11厘米长的肠段，于近端和远端插管，并分别在肠系膜静脉和肠系膜上动脉插管，建立大鼠原位小肠灌流模型，通过肠道灌流50 µmol/kg的芹菜素、大黄素、大黄酚，于灌流后10、20、30、40、50、60 min，分别收集肠道和肠系膜静脉的流出液。采用一级全扫描质谱分析和二级质谱分析（子离子扫描），鉴定肠道和血管流出液中多酚类化合物的代谢产物。用β-葡萄糖醛酸酶和硫酸酯酶处理样品，采用高效液相色谱-质谱连用的方法，定量分析原形药物、葡萄糖醛酸化和硫酸酯化代谢产物浓度的变化规律。（3）24孔板培养Caco-2细胞，密度为5×104个/孔，至14-20天时，用于测定多酚类药物在Caco-2细胞中的摄取特点。实验前换无血清培养液37℃培养24h，用HBSS液平衡15分钟，将待测的多酚类药物按设定浓度和Caco-2细胞孵育一定的时间（5、10、30、60、120 min），同时选择不同的转运载体抑制剂，考察细胞摄取药物浓度与载体之间的关系。液氮反复冻融、高速离心裂解细胞，乙酸乙酯提取细胞裂解液中的药物，通过高效液相色谱法和β-葡萄糖醛酸酶、硫酸酯酶水解的方法，定量分析原形药物、葡萄糖醛酸化和硫酸酯化代谢产物的浓度，并通过考马斯亮蓝法测定细胞中蛋白含量。接种Caco-2细胞于Millicell培养皿上，密度为1×105个/孔，细胞培养至19-21天时， Millicell-ERS 电压电阻仪测定Millicell培养皿两侧的电阻值（TEER），评价Caco-2细胞单层膜的完整性，当TEER值≥300 ·cm2时，可用于测定多酚类药物在Caco-2细胞中的转运特点。实验前换无血清培养液37℃培养24h，用HBSS液平衡15分钟，分别从顶侧及基底侧加入1.5mL和2.0mL一定浓度的多酚类药物，按照设定的时间点（10、20、30、40、50、60 min）分别从顶侧及基底侧取样，加入200μL内标溶液，混匀，高速离心，用β-葡萄糖醛酸酶和硫酸酯酶处理样品，通过高效液相色谱法定量分析原形药物、葡萄糖醛酸化和硫酸酯化代谢产物浓度的规律。
结果 （1）大鼠灌胃给予芹菜素、大黄酚、大黄素和白藜芦醇后，药物可被迅速吸收进入血液循环，芹菜素、大黄素和白藜芦醇的吸收速度均比较快，血药浓度时间-曲线呈明显的双峰现象。芹菜素的峰值血药浓度为45±2ng/mL，达峰时间为0.5小时，给药后2小时出现第二个吸收高峰，峰值为 34±1ng/mL。大黄素在给药0.5小时后达到吸收高峰，血药浓度为72 ± 6ng/mL，与芹菜素相比，其第二个吸收高峰出现的较晚，大约在给药后8小时出现第二个吸收峰，其血药浓度峰值为 49±6ng/mL。白藜芦醇吸收迅速，在给药0.2小时就出现第一个吸收高峰，峰值血药浓度为94±5ng/mL，给药后8小时出现第二个吸收高峰，血药浓度为 18±2ng/mL。与前三者相比，大黄酚的吸收量较多，但吸收速度相对比较缓慢，2小时达到高峰，血药浓度为326±14ng/mL，未观察到双峰现象。大鼠灌胃给予50 µmol/kg芹菜素、大黄酚、大黄素、白藜芦醇，其AUC分别为659、1721、809、98 (ng/ml)*h，T1/2Ke分别为2.112、8、2.931、1.823 h。对血浆样品提取物进行一级全扫描质谱分析，可检测到特定的准分子离子峰（m/z 349.7和m/z 446，m/z 333.7和m/z 429.3，349.7和 m/z 446，m/z 306.9和 m/z 402.7），分别比原形药物（芹菜素、大黄酚、大黄素和白藜芦醇）的准分子离子峰多80 u和176u，选择性对这些离子峰进行二级质谱分析（子离子扫描），发现它们均能脱去176 u和80u的碎片，正好对应于代谢产物中的葡萄糖醛酸和硫酸酯部分。分别用β-葡萄糖醛酸酶和硫酸酯酶处理样品，经MRM方式检测，发现芹菜素（m/z 268.9→116.8）、大黄酚（m/z 253.3→225.2）、大黄素（m/z 269.4→225.4 ）和白藜芦醇（m/z 226.9→142.8）的色谱峰面积均高于未加酶组样品的峰面积，提示上述药物代谢过程中均有葡萄糖醛酸化和硫酸酯化代谢产物的生成。在大鼠体内葡萄糖醛酸化芹菜素的量相对较多，在给药后1小时出现代谢高峰，葡萄糖醛酸化芹菜素的峰浓度为51±3 ng/mL。而硫酸酯化芹菜素量较少，其在给药后0.5 - 4小时之间量较高。体内葡萄糖醛酸化大黄酚的量在8小时出现高峰，峰浓度为82±9 ng/mL，占血中药物（大黄酚+葡萄糖醛酸化大黄酚+硫酸酯化大黄酚）总量的51.7%，而硫酸酯化大黄酚仅占血中药物（大黄酚+葡萄糖醛酸化大黄酚+硫酸酯化大黄酚）总量的2.5%。葡萄糖醛酸化大黄素在给药后1 -12 小时之间生成的量较多，硫酸酯化大黄素在给药后0.5小时出现高峰，占血中药物（大黄素+葡萄糖醛酸化大黄素+硫酸酯化大黄素）总量的15.22 %。葡萄糖醛酸化白藜芦醇在0.5小时出现高峰，浓度为113 ± 9ng/mL，占血中药物（白藜芦醇+葡萄糖醛酸化白藜芦醇+硫酸酯化白藜芦醇）总量的42.54 %，而硫酸酯化的白藜芦醇占血中药物（白藜芦醇+葡萄糖醛酸化白藜芦醇+硫酸酯化白藜芦醇）总量的17.9%。（2）对灌流液提取物进行一级全扫描质谱分析，可检测到特定的准分子离子峰，分别比原形药物（芹菜素、大黄酚和大黄素）的准分子离子峰多80 u和176u。选择性对这些准分子离子峰进行二级质谱分析（子离子扫描），发现它们均能脱去176 u和80u的碎片，正好对应于代谢产物中的葡萄糖醛酸和硫酸酯部分。分别用β-葡萄糖醛酸酶和硫酸酯酶处理样品，经MRM方式检测，发现芹菜素、大黄酚和大黄素的色谱峰面积均高于未加酶组样品的峰面积，提示葡萄糖醛酸化和硫酸酯化的代谢产物分别经β-葡萄糖醛酸酶和硫酸酯酶水解后释放出原形的药物。实验结果发现，大约28.88%的芹菜素出现在血管的一侧，其中原形药物占16.39%，葡萄糖醛酸化芹菜素和硫酸酯化芹菜素分别占8.7%和3.79%，肠道流出灌流液中存在葡萄糖醛酸化芹菜素和硫酸酯化芹菜素，分别为灌流药物总量的12.93%和1.83%。约48.56%的大黄酚出现在血管的一侧，其中原形药物占32.22%，葡萄糖醛酸化大黄酚和硫酸酯化大黄酚分别占13.85%和 2.48 %。肠道流出灌流液中存在葡萄糖醛酸化大黄酚和硫酸酯化大黄酚，分别为灌流药物总量的 8.63%和4.34%。大约22.55%的大黄素出现在血管的一侧，其中原形药物占12.01%，葡萄糖醛酸化大黄素和硫酸酯化大黄素分别为8.69%和1.84%。肠道流出灌流液中存在葡萄糖醛酸化大黄酚和硫酸酯化大黄酚，分别为灌流药物总量的5.23%和1.04%。芹菜素、大黄酚、大黄素和它们代谢产物的总回收率分别为98.09 ± 3.5%，99.53 ± 4.3%，和96.49± 2.2%。与芹菜素和大黄素相比，大黄酚的吸收量较多，48.56%的灌流药物以大黄酚、葡萄糖醛酸化大黄酚和硫酸酯化大黄酚三种形式转运到血管侧，而芹菜素和大黄素转运到血管侧的比例较少，分别为28.88% 和22.55%；但是，芹菜素和大黄素进入血管侧的代谢产物较多，分别为43.23%和46.72%，而大黄酚进入血管侧的代谢产物所占比例相对较少，为33.53%，大部分以原形的形式入血。（3）芹菜素、大黄酚、大黄素和白藜芦醇均可被Caco-2细胞迅速摄取，大黄酚和大黄素的最大摄取浓度为414±15nmol/L·mg·protein 和108±12nmol/L·mg·protein。多酚类化合物在Caco-2细胞中的摄取随着药物浓度的增加呈现一定的差别，芹菜素和大黄素的饱和浓度为50 µM，白藜芦醇的饱和浓度为100 µM，而大黄酚在Caco-2细胞中的摄取量随着药物浓度的加大而增长，200 µM时仍未达到饱和。药物与Caco-2细胞孵育1小时后，细胞中大黄素和白藜芦醇代谢产物的量均超过了药物总量（原形药物+葡萄糖醛酸化+硫酸酯化代谢产物的总量）的50%，其中葡萄糖醛酸化和硫酸酯化白藜芦醇分别占26.56%和25.16%，而大黄素的这两种代谢产物所占的比例分别为45.16%和10.75%。芹菜素和大黄酚在细胞中代谢产物的量稍低于前两者，其中葡萄糖醛酸化芹菜素和硫酸酯化芹菜素分别为17.82%和13.27%，而大黄酚的这两种代谢产物所占的比例分别为33.45%和6.7%。根皮苷可以抑制Caco-2细胞对大黄酚和大黄素的摄取，细胞中药物的浓度分别下降了41%和34%，而对芹菜素和白藜芦醇的摄取没有明显影响。P糖蛋白抑制剂维拉帕米、MRP2抑制剂环苞菌素A能够增加多酚类化合物在Caco-2细胞中的摄取量。
Absorption and metabolism of polyphenols in the intestine
Postgraduate： Teng Zenghui
Supervisor： Mei Qibing
Assitant Supervisor： Zhou Siyuan
Department of Pharmacology，School of Pharmacy，Fourth Military Medical University，Xi’an 710032，China
Purpose Polyphenols, a group of complex naturally occurring compounds, are widely distributed throughout the plant kingdom and thus are readily consumed by humans. As a member of polyphenols family, dietary anthraquinone have received much attention as potential protectors against a variety of human diseases, in particular cardiovascular disease and cancer. Epidemiological evidence has long suggested that dietary polyphenols, which are abundant in fruits and vegetables, can reduce the risk of cancer. Such diets are well known to contain a variety of chemicals that can affect the carcinogenic process in many ways. A large number of mechanisms of action have been investigated, including antioxidant properties and effects on enzymes and signal transduction pathways. Increasing evidence for the possible effects of plant polyphenols on human health have been obtained in in vitro and in vivo systems. Intestinal absorption is a prerequisite for a possible causal relationship between polyphenols intake and its proposed chemopreventive action. However, data on their absorption from the gastrointestinal tract are still scarce. To address this issue, we analysed the pharmacokinetics of polyphenols in rat plasma and evaluated the contribution of the small intestine to the absorption and first-pass metabolism of polyphenols using an isolated rat small intestine perfusion model, the human Caco-2 cell model.
Methods (1) The selected polyphenols, including apigenin, chrysophanol, emodin and resveratrol, were suspended in 0.5% CMC-Na and administered at a dose of 50 µmol/kg to rats in experiments involving oral administration. Blood samples were collected from the vena abdominalis under light ether anesthesia at different times(0.08、0.17、0.5、1、2、4、8、12、16、24、36h). The samples were immediately centrifuged for 10 min at 3000 rpm to obtain plasma, which was kept frozen at -70 °C until analysis. We developed a high-throughput and sensitive bioanalytical method using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) equipped with an electrospray ionization interface used to generate negative ions [M-H]– for the estimation of the concentration of the selected polyphenols and their metabolites. Quantitation was performed by multiple reaction monitoring (MRM) of the deprotonated precursor ion and the related product ion for the selected polyphenols after treatment with the β-glucuronidase and sulfatase, respectively. Pharmacokinetic parameters of different polyphenols and their metabolites were calculated by the 3p97 software after oral administration of the drugs to rat. (2) The rats were anesthetized with an intra-abdominal injection of a mixture containing 40 mg/kg phenobarbital sodium. The animals were then heparinized (90 U/ kg–1) via the vena caudalis. A 7- to 11-cm-long segment of the intestine was identified and separated. Silicone tubing was placed inside both ends of the segment, and the tube at the proximal side was connected to a peristaltic pump for luminal perfusion. A polyethylene cannula, connected to a peristaltic pump for the vascular perfusion, was inserted into the superior mesenteric artery. The solutions on both sides were circulated by gas lift with 95% O2 and 5% CO2 throughout the transport studies. Samples obtained from the outlet of the mesenteric vein and luminal aliquots were collected into the preweighted microtubes every 10 min and stored at -70°C until analysis. We developed a high-throughput and sensitive bioanalytical method using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) equipped with an electrospray ionization interface used to generate negative ions [M-H]– for the estimation of the concentration of the selected polyphenols and their metabolites. Quantitation was performed by multiple reaction monitoring (MRM) of the deprotonated precursor ion and the related product ion for the selected polyphenols after treatment with the β-glucuronidase and sulfatase, respectively. (3) For all cellular uptake studies of polyphenols, Caco-2 cells were seeded at a cell density of 5×104cells/cm2 on twenty-four well plastic plates. Cells were used 14 to 21 days after seeding. Fresh culture medium was replaced 24 h before uptake experiments. The selected polyphenols at various concentrations was added to evaluate the uptake characteristics at different times(5、10、30、60、120 min). To determine the intracellular polyphenols concentration, the cell lysate was obtained by subjecting the drug-containing cells to three freeze-thaw cycles in liquid nitrogen. Protein concentrations were measured by the method of Bradford with bovine serum albumin as a standard. For transport experiments, Caco-2 cells were seeded at a cell density of 1×105cells/cm2 on Millicells. Cells were used 19 to 21 days after seeding and the value of TEER should more than 300 ·cm2. Fresh culture medium was replaced 24 h before transport experiments. The selected polyphenols at various concentrations was added from the apical or basolateral side to evaluate the transport characteristics at different times(5、10、30、60、120 min). The samples were analyzed by reverse phase HPLC system after treatment with the β-glucuronidase and sulfatase, respectively.
Results (1) Following oral administration apigenin, chrysophanol, emodin and resveratrol, the selected polyphanols can be absorbed rapidly, and there are obvious double peaks phenomenon in the CT curve of apigenin, emodin and resveratrol. The maximum concentration the selected polyphanols were 45 ±2 ng/mL, 326±14ng/mL, 72±6ng/mL and 94±5ng/mL, respectively. And the AUC and the elimination half-time were 659、1721、809、98 (ng/ml)*h and 4.1、8.0、2.9、1.8h for apigenin, chrysophanol, emodin and resveratrol, respectively. Glucuronide and sulfate were identified and quantified by enzymatic hydrolysis with glucuronidase and sulfatase using LC/MS/MS. After the full scan model, the special ion peak were detected, which have epactal fragment than the original drugs with 80 u and 176u. We presume that these fragment were the glucuronidated and sulfated parts. The selected polyphanol conjugates were quantified based on original drug concentration after enzymatic incubation of luminal and vascular effluents. Quantitation was performed by multiple reaction monitoring (MRM) of the deprotonated precursor ion and the related product ion for the selected polyphenols after treatment with the β-glucuronidase and sulfatase, respectively. We found the chromatographic peak area were more than the control group. The plasma level of apigenin glucuronide was higher than that of apigenin sulfate, the plasma levels of apigenin glucuronide reached Cmax of 51±3ng/mL within 1 hour. The plasma levels of resveratrol glucuronide rapidly reached Cmax within 0.5 hour. (2) Glucuronide and sulfate were identified and quantified by enzymatic hydrolysis with glucuronidase and sulfatase using LC/MS/MS. After the full scan model, the special ion peak were detected, which have epactal fragment than the original drugs with 80 u and 176u. We presume that these fragment were the glucuronidated and sulfated parts. The selected polyphanol conjugates were quantified based on original drug concentration after enzymatic incubation of luminal and vascular effluents. Quantitation was performed by multiple reaction monitoring (MRM) of the deprotonated precursor ion and the related product ion for the selected polyphenols after treatment with the β-glucuronidase and sulfatase, respectively. We found the chromatographic peak area were more than the control group. Approximately 28.88% of apigenin that was administered appeared at the vascular side, chiefly as free rutin (16.39%), but also as emodin glucuronide (8.7%) and emodin sulfate (3.79%). The main compound in the luminal effluent was apigenin, accompanied by lesser amounts of apigenin glucuronide and apigenin sulfate. Apigenin glucuronide (12.93%) and apigenin sulfate (1.83%) were found at the luminal side. For chrysophanol, the percentage of chrysopahol glucuronide and sulfate were 13.85% and 2.48 % at the vascular side. Approximately 22.55% of emodin that was administered appeared at the vascular side, chiefly as free emodin (12.01%), but also as emodin glucuronide (8.69%) and emodin sulfate (1.84%). The main compound in the luminal effluent was emodin, accompanied by lesser amounts of emodin glucuronide and emodin sulfate. Emodin glucuronide (5.23%) and emodin sulfate (1.04%) were found at the luminal side. The mean total recovery of emodin and metabolites was 96.49± 2.2%. In control perfusion experiments with the selected polyphanols-free basic perfusion media, no polyphanols or its conjugates were detected. (3) The selected polyphenols can be absorpted rapidly by the caco-2 cells, and the maximal concentration of chrysophanol and emodin were 414±15 and 108±12 nmol/L·mg·protein, respectively. When the concentration of emodin and apigenin were increased from 2 mM to 50 mM, the intracellular concentration increased almost linearly. However, the saturation was apparent when the concentration of was increased from 50 mM to 200 mM. The chrysophanol uptake continued to increase when the concentration of chrysophanol was increased from 2 mM to 200 mM, and no saturation was apparent. And the saturation was 100 mM for resveratrol. After the incubation with the Caco-2 cells for 1 hour, the selected polyphenols were metabolized by the phaseⅡenzyme extensively, the percentage of glucuronidated and sulfated resveratrol were 26.56% and 25.16%, respectively. The glucuronidated and sulfated emodin were 45.16% and 10.75%. For apigenin, the percentage of metabolites were 17.82% and 13.27%. The cellular uptake of emodin and chrysophanol were reduced by approximately 34% and 41% in the presence of 50 µM phloridzin, but have no obvious effect on the cellular uptake of apigenin and resveratrol. Verapamil and cyclosporine can increase the uptake of the selected polyohanols in caco-2 cells.
Conclusions （1）The selected polyphenols can be absorpted into blood circulation rapidly after oral administration.（2）The bioavailability of these selected polyphenols was poor after oral administration.（3）Apigenin, chrysophanol, emodin and resveratrol can be metabolized by the phaseⅡenzyme in rat intestine. One parts of the glucuronide or sulfate polyphenols were absorpted into blood circulation, the another were effluxed to the luminar side. The first metabolism may be the reason for the poor bioavailability in rat intestine.（4） The glucuronide chrysophanol or emodin was higher than sulfate metabolites in caco-2 cells. There are no obvious difference between glucuronide apigenin or resveratrol and their sulfate metabolites.（5） Phloridzin can reduced the cellular uptake of emodin and chrysophanol in caco-2 cells, which suggested that emodin and chrysophanol can be absorbed through the way of SGLT1.（6）Cyclosporine can increase the uptake of the selected polyohanols in caco-2 cells, which means MRP2 was the transport way for these compound.（7） The phenolic hydroxyl group of these polyphenols was related to their absorption and metabolism in the intestine possibly. The different transporters and phaseⅡenzyme can regulate the process and affect their pharmacological effect.
Key words polyphenols; pharmacokinetics; intestine; transport; metabolism; small intestine perfusion model; caco-2 cell; HPLC; MS