摘要：目的 探索与人类代谢柠檬苦素相近的实验动物物种。方法 代谢组用人、狗、猪、猴、兔、大鼠、小鼠7种肝微粒体分别与柠檬苦素孵育。对照组用灭活的肝微粒体与柠檬苦素孵育。空白组不加柠檬苦素。用高效液相紫外检测器检测柠檬苦素标准品溶液和上述样品，观察代谢组样品有无代谢产物。通过酶动力学实验结果计算不同种属肝微粒体酶对柠檬苦素的清除率。用高效液相高分辨质谱检测器检测上述样品，推测代谢产物结构。结果 紫外检测色谱图发现人、狗、猪、兔、大鼠、小鼠共同代谢产物峰M1，保留时间为13.3 min。大鼠还有两个代谢产物峰（M2为10.6 min、、M3为8.6 min）。猴的代谢产物峰保留时间为8.1 min和10.1 min（M4和M5）。柠檬苦素在人、狗、猪、大鼠、小鼠肝微粒体中的清除分别为0.608、0.454、0.501、4.249、1.831 μL/(min·mg)。高分辨质谱检测结果推测M1为柠檬苦素的C-16位羟基化化合物。结论 猪、狗、大鼠、小鼠清除率与人类接近。推测柠檬苦素的人肝微粒体酶主要代谢产物是其16位碳羟基化化合物。

Abstract: Objective: To explore which species experimental animal are similar to human about the metabolism of limonin. Methods: Limonin was incubated separately with liver microsomes,such as the humans liver microsomes, the dogs liver microsomes, the pigs liver microsomes, the monkeys liver microsomes, the rabbits liver microsomes, the rats liver microsomes and the mices liver microsomes, that were as experimental groups. In contrast, limonin was incubated with inactivated liver microsomal enzymes served as control groups. And the limonin were not added to the blank group. All above samples and limonin standard solution were detected by HPLC-UV detector to observe the metabolism of limonin.According to the results of enzyme kinetics experiments, the clearance rate of limonin by liver microsomal enzymes of different species was calculated.The above samples were detected by high performance liquid chromatography-high resolution mass spectrometry (HPLC-MS) detector to infer the metabolite structure.Results: M1 peak was found in human, dog, pig, rabbit, rat and mouse by UV detection chromatogram, and the retention time was 13.3 minutes. In addition, rats had two metabolite peaks (M2:10.6 min, M3:8.6 min). However, it was interesting that the metabolite peaks in monkeys were different from those in humans, with retention times of 8.1 min and 10.1 min (M4 and M5). The clearance of limonin in human, dog, pig, rat and mouse liver microsomes was 0.608, 0.454, 0.501, 4.249 and 1.831 μL/(min·mg), respectively. And the results of high resolution mass spectrometry suggested that M1 was a hydroxyl compound at C-16 position.Conclusion: The clearance rate of pigs, dogs, rats and mice is close to that of human. It is speculated that the main metabolite of limonin catalyzed by liver microsomal enzymes is its 16-carbon hydroxyl compound.

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辽宁省教育厅基础研究基金(JYTJCZR2020055)；辽宁省博士基金(2019-BS-100)；辽宁省振兴人才计划(XLYC2007142)；技术发展任务(2021003)