Keywords: Rhubarb; anthraquinone; pharmacokinetics; ulcerative colitis; intestinal bacteria; metabolism; glucuronidation. Background: Ulcerative colitis is an inflammatory disease in the colonic mucosa and submucosa with an unknown etiology. Ulcerative colitis relapses easily and it is difficult to cure. Imbalance of intestinal bacteria is considered an important factor in the pathogenesis of ulcerative colitis. As a famous traditional Chinese medicine, rhubarb obtains good effect in the treatment of ulcerative colitis clinically. Anthraquinone is the most important polyphenolic compounds in Rhubarb. In vivo pharmacokinetics of Rhubarb anthraquinones in rat or human have been reported for many times. Glucuronide conjugates were believed to be the main form of anthraquinones in body. However, there are no reports on the pharmacokinetics of Rhubarb anthraquinones in the pathological state of ulcerative colitis. In present study, we aimed rats to study in vivo pharmacokinetic profiles of rhubarb anthraquinone, the changes of intestinal bacterial metabolism and intestinal bacteria, in the ulcerative colitis, compared to the normal rats, and we tried to discuss the links among them. Finally, in vitro glucuronidation of anthraquinones were researched in order to explain the in vivo pharmacokinetics of anthraquinones, and the structrue and metabolism relationship was disscussed in glucuronidation. Methods: Dextran sulfate sodium was used to induce ulcerative colitis in SD rats. In vivo pharmacokinetic characterization of anthraquinones were studied in the pathological mode. The metabolism ability towards anthraquinone glycosides was studied in the intestinal bacteria metabolism model. The changes of intestinal bacteria were investigated by real-time PCR technology. In vitro glucuronidation of anthraquinones were researched in order to explain the in vivo pharmacokinetics of anthraquinones. Results: Pharmacokinetic studies showed that glucuronide conjugates were the main form of anthraquinones in vivo. In ulcerative colitis rats, AUC and T1/2 of anthraquinones changed after hydrolysis, so did the ratio of AUC between before hydrolysis and after hydrolysis. In the intestinal bacterial metabolism experiments, the activity catalyzing anthraquinone glucosides to aglycones of intestinal bacteria was lower in the pathological mode, compared to the healthy rats. However, the more aglycones were transformed to other compounds in UC rats. As well as C.leptum, the probiotic Bifidobacterium and and Lactobacillus decreased, however, B.fragilis incrased significantly in the pathological model. When the model is done, C.coccoides and Enterobacteriaceae in UC rats almost equied to that in the healthy rats. In vitro glucuronidation studies found that rhubarb anthraquinones except rhein underwent intense glucuronidation. The elimination rate and positional preference of glucuronidation varied with substituted position and moiety. UGT 1A catalyzed glucuronidation of most anthraquinones at varied activity and positional preference. UGT 1A9 showed the highest activies at most cases. Conclusion: In ulcerative colitis rats, the decrease of Bifidobacterium and Lactobacillus, and the changes of other bacteria, resulted in lower anthraquinone aglycones exposure to the intestinal, which might be a important reason why the pharmacokinetic parameters of anthraquinone changed. Glucuronide conjugates were the main pattern of anthraquinones in vivo due to the high glucuronidation activity of anthraquinones that was confirmed.

关键词：大黄；蒽醌；药代动力学；溃疡性结肠炎；肠道菌群；代谢；葡萄糖醛酸化 研究背景：作为中国传统著名中药，大黄在临床上具有广泛的应用，其蒽醌成分被认为是 大黄发挥治疗作用的主要活性成分。研究表明，大黄蒽醌口服后主要经肠道菌群水解后吸 收，在正常动物/人体内主要以结合型（葡萄糖醛酸和/或磺酸）存在，提示肠道菌群组成/ 功能以及肝脏、肠道 II 相代谢能力的改变均可能影响大黄的体内处置，从而影响其治疗效 应。本研究旨在通过比较大黄主要蒽醌类化合物（大黄素、大黄酸、大黄酚、大黄素甲醚、 芦荟大黄素）在溃疡性结肠炎（ulcerative colitis，UC）模型大鼠以及正常大鼠体内的药代 动力学，阐明大黄蒽醌在 UC 状态下体内处置的变化及影响机制；此外，本研究对大黄蒽 醌类化合物葡萄糖醛酸结合的结构-代谢关系以及主要代谢酶亚型进行探讨，为以大黄为主 的复方配伍和临床中西药联用提供依据。 方法：采用 5%葡聚糖硫酸纳饮水 7 天诱导加 3%葡聚糖硫酸纳饮水 14 天维持的方法建立大 鼠溃疡性结肠炎模型，以正常大鼠平行饮水作为对照组，研究 UC 病理状态对大黄蒽醌化 合物体内药代动力学的影响；利用肠内菌群体外代谢模型比较 UC 大鼠肠道细菌代谢蒽醌 糖苷能力的改变，用基于 16S rRNA 的荧光实时定量 PCR 分析 UC 模型大鼠肠道细菌组成 的变化，大黄蒽醌葡萄糖醛酸化研究采用体外温孵肠、肝微粒体以及重组人 UGT 亚型。大 黄蒽醌的定性定量分析采用酸水解结合 HPLC-UV 和/或 HPLC-MS/MS 分析，代谢产物的鉴 定采用 HPLC-MS/MS 以及 NMR 分析，药动学数据分析使用 WinNonlin5.2 软件。 结果：口服给予大黄提取物后，大黄蒽醌在 UC 及正常大鼠体内均主要以二相代谢结合物 存在；与正常大鼠相比，UC 大鼠血浆中，各蒽醌化合物（结合型+游离型）的 AUC，t1/2 以及其蒽醌苷元（游离型）的 AUC 百分比发生改变；相应地，UC 大鼠的肠道菌群代谢蒽 醌糖苷的能力降低，而 UC 大鼠肠微粒体对蒽醌苷元的葡萄糖醛酸化程度提高， UC 大鼠 肝微粒体对蒽醌苷元的代谢程度未见明显改变。RT-PCR 分析大鼠肠道细菌的结果显示， 益生菌 Bifidobacterium 和 Lactobacillus 含量减少，C.leptum 呈现同样下降趋势，而 B.fragilis 含量呈明显增加，在模型完成时，C.coccoides 和 Enterobacteriaceae 的含量没有明显的区别。 对大黄蒽醌类化合物葡萄糖醛酸化进一步研究发现，除大黄酸外，其他大黄蒽醌类化合物 均发生较强的葡萄糖醛酸结合反应。由于取代基不同/取代位点不同，蒽醌化合物葡萄糖醛 酸结合的清除速率以及位点选择性各异，其中 C-3 羟基（大黄素）是大黄蒽醌发生葡萄糖 醛酸化的优势位点；蒽醌化合物的葡萄糖醛酸结合反应主要由 UGT 1A 亚家族催化，其中 以 UGT 1A9 活性最高。 结论：在 UC 状态下，大鼠肠道菌群中表达-葡萄糖醛酸苷酶的 Bifidobacterium 和 Lactobacillus 的量减小，其他细菌的量也发生变化，使得 UC 大鼠肠道菌群对大黄蒽醌结合 型的转化降低，蒽醌苷元的生成量减少，可能是导致大黄蒽醌在 UC 大鼠体内系统暴露水 平降低的主要原因；UC 大鼠肠道微粒体对大黄蒽醌的葡萄糖醛酸化活性明显升高，可能 是影响蒽醌类化合物在大鼠体内主要存在形式和系统暴露水平的另一主要因素。此外，大 黄蒽醌类化合物在体内主要由 UGT1A 代谢酶亚家族介导发生较强的葡萄糖醛酸结合反应， 可能影响其复方配伍疗效和中西药联用临床安全性。