Li Qiang, Wang Liping, Dai Peimin, Zeng Xuejun, Qi Xiaoxiao, Zhu Lijun, Yan Tongmeng, Wang Ying, Lu Linlin, Hu Ming, Wang Xinchun, Liu Zhongqiu
School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, China; International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, China.
The First Affiliated Hospital of the Medical College, Shihezi University, Shihezi, China.
J Chromatogr A. 2015 May 22;1395:116-28. doi: 10.1016/j.chroma.2015.03.069. Epub 2015 Apr 1.
The use of dietary flavones is becoming increasingly popular for their prevention of cancers, cardiovascular diseases, and other diseases. Despite many pharmacokinetic studies on flavone mixtures, the position(s) of glucuronidation sites on the flavone skeleton in vivo remain(s) uncertain because of the lack of a convenient method to differentiate the isomers in biological samples. Accordingly, this study aimed to develop a new strategy to identify the position of the mono-O-glucuronide of flavones in vivo and to simultaneously determine the parent agent and its major metabolites responsible for complex pharmacokinetic characteristics. The novel strategy involves accurate mass measurements of flavone glucuronides, their Co(II) (flavone glucuronide-H) (4,7-diphenyl-1,10-phenanthroline)2 complexes generated via the post-column addition of CoBr2 and 4,7-diphenyl-1,10-phenanthroline, and their mass spectrometric fragmentation by UPLC-DAD-Q-TOF and the comparison of retention times with biosynthesized standards of different isomers that were identified by analyzing the shift in UV spectra compared with the spectra of their respective aglycones. We successfully generated a metabolite profiling of flavones in rat plasma after oral administration of a flavone mixture from Dracocephalum moldavica L., which was used here as the model to demonstrate the strategy. Twelve flavone glucuronides, which were glucuronidated derivatives of acacetin, apigenin, luteolin, diosmetin, chrysoeriol and cirsimaritin, were detected and identified. Glucuronidation of the flavone skeleton at the 3'-/7-position was more prevalent, however, luteolin 4'-glucuronide levels exceeded luteolin 7-glucuronide levels. Based on the UDP-glucuronosyltransferase (UGT) metabolism profiling of flavones in rat plasma, six main compounds (tilianin, acacetin 7-glucuronide, apigenin 7-glucuronide, luteolin 3'-glucuronide, acacetin, and apigenin) were selected as pharmacokinetic markers. Pharmacokinetic results indicated that their maximal concentrations in blood were obtained within 0.4h, except for the concentration of luteolin 3'-glucronide (approximately 9h). Rat exposure was practically non-linear under the studied dosages (200 to 400mg/kg).
膳食黄酮因其对癌症、心血管疾病及其他疾病的预防作用而越来越受到人们的青睐。尽管对黄酮混合物进行了许多药代动力学研究，但由于缺乏一种在生物样品中区分异构体的便捷方法，黄酮骨架上葡萄糖醛酸化位点在体内的位置仍不明确。因此，本研究旨在开发一种新策略，以确定黄酮单 -O- 葡萄糖醛酸苷在体内的位置，并同时测定母体药物及其主要代谢产物，这些代谢产物决定了复杂的药代动力学特征。该新策略包括对黄酮葡萄糖醛酸苷进行精确质量测量，通过柱后添加 CoBr₂ 和 4,7 - 二苯基 -1,10 - 菲咯啉生成其 Co(II)(黄酮葡萄糖醛酸苷 -H)(4,7 - 二苯基 -1,10 - 菲咯啉)₂ 配合物，并通过超高效液相色谱 - 二极管阵列 - 四极杆飞行时间质谱联用仪对其进行质谱裂解，以及将保留时间与通过分析与各自苷元光谱相比的紫外光谱变化而鉴定的不同异构体的生物合成标准品进行比较。我们成功地生成了大鼠口服来自 Moldavica 龙蒿（Dracocephalum moldavica L.）的黄酮混合物后血浆中黄酮的代谢物谱，在此将其用作模型来证明该策略。检测并鉴定了 12 种黄酮葡萄糖醛酸苷，它们是刺槐素、芹菜素、木犀草素、香叶木素、 Chrysoeriol 和 cirsimaritin 的葡萄糖醛酸化衍生物。黄酮骨架在 3'-/7 - 位的葡萄糖醛酸化更为普遍，然而，木犀草素 4'- 葡萄糖醛酸苷的水平超过了木犀草素 7 - 葡萄糖醛酸苷的水平。基于大鼠血浆中黄酮的尿苷二磷酸葡萄糖醛酸基转移酶（UGT）代谢谱，选择了六种主要化合物（田蓟苷、刺槐素 7 - 葡萄糖醛酸苷、芹菜素 7 - 葡萄糖醛酸苷、木犀草素 3'- 葡萄糖醛酸苷、刺槐素和芹菜素）作为药代动力学标志物。药代动力学结果表明，除木犀草素 3'- 葡萄糖醛酸苷的浓度（约 9 小时）外，它们在血液中的最大浓度在 0.4 小时内达到。在所研究的剂量（200 至 400mg/kg）下，大鼠的暴露实际上是非线性的。
