柠檬苦素体外代谢的种属差异

IN VITRO METABOLIC DIFFERENCES OF LIMONIN IN DIFFERENT SPECIES

目的研究柠檬苦素代谢的种属差异,为柠檬苦素体内代谢研究提供实验动物选择的依据。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峰。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代谢产物,推测代谢产物结构及反应类型。结果HPLC图谱显示人、犬、猪、兔、大鼠、小鼠有共同的代谢产物峰M1(RT13.3min);大鼠还有两个代谢产物峰M2(RT10.6min)和M3(RT8.6 min);猴的代谢产物峰为M4(RT8.1 min)和M5(RT10.1 min)。柠檬苦素在人、犬、猪、大鼠、小鼠肝微粒体中的清除分别为0.608、0.454、0.501、4.249、1.831μL/(min·mg)。高分辨质谱检测结果推测M1为柠檬苦素的C-16位羰基被还原为羟基的化合物。结论猪、犬、大鼠、小鼠清除率与人类接近。我们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。

Objective To study which species experimental animal are similar to human about the metabolism of limonin,and it will be a basis for the selection of experimental animals for the metabolism of limonin in vivo.Methods Limonin was incu-bated separately with liver microsomes,such as the humans liver microsomes,the dogs liver microsomes,the pigs liver micro-somes,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 con-trol 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 clear-ance 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 me-tabolite 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 1.831μL/(min·mg),respectively.And the results of high resolution mass spectrometry suggested that M1 was a hydroxyl compound at C-16 position.Conclusions 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.