大鼠灌胃缩泉方制剂后尿、粪中的益智活性成分及代谢物的测定

Identification of known chemicals and their metabolites from Suoquan formula in rat urine and feces

目的研究大鼠灌胃缩泉胶囊和缩泉丸后,诺卡酮、益智黄酮(杨芽黄素、伊砂黄素、白杨素、山奈素和芹菜素-4',7-二甲氧醚)和益智中二芳基庚烷类成分(益智酮甲、益智酮乙、益智醇)在尿和粪中的物质形式。方法雄性Sprague Dawley经灌胃给予缩泉胶囊(3.54 g/kg)和缩泉丸(3.77 g/kg)后置于代谢鼠笼中,收集12 h的尿液和粪球。粪球称重后加入9倍体积的去离子水,高速匀浆。分别取100μl尿液和粪球匀浆液于离心管中,加入300μl甲醇,漩涡振摇5 min,13 000次/min下离心10 min,取上清液,在N2下吹干,残渣用50μl甲醇复溶,再次振摇和离心,取上清液40μl,利用液相色谱-串联质谱联用仪(LC-MS/MS)进行分析。结果根据上述对照品在生物样品中的LC-MS/MS出峰情况,通过比较空白尿、粪样品与给药后相应的尿粪样品,研究发现,大鼠灌胃后,在尿液和粪便中主要检测到原形物质形式,未检测到葡萄糖醛酸结合物等二相代谢产物。结论大鼠尿液中检测到的诺卡酮、益智酮甲以及杨芽黄素等成分,可能是直接作用于膀胱而发挥调节膀胱功能的活性成分。

Objective To identify the flavonoids（tectochrysin, izalpinin, chrysin, kaempferide and apigenin-4＇,7-dimethylether） and their metabolites, as well as diarylheptanoids（yakuchinone A, yakuchinone B and oxyphyllacinol） and their metabolites, in rat urine and feces after oral administration of Suoquan capsules or pills.Methods After male Sprague Dawleyoral rats were administratered Suoquan pills（3.54 g/kg） and Suoquan capsules（3.77 g/kg）, they were placed into the metabolism of the cage. Their urine and dung were collected 12 h later. The dung was weighed and added 9 volumes of deionized water and then homogenized at high speed divided method. 100 μl homogenate of urine and dung were taken into centrifuge tube, with 300 μl methanol added, shaked 5 min by vortex and centrifuged 10 min at 13 000 r/min. The supernatant were drawed and dried under N2. The residue was reconstituted using 50 μl methanol liquid, shaked and centrifugated again. 40 μl supernatant was drawed and analyzed by liguid chromatography-tandem mass spectrometry（LC-MS/MS）. Results According to the peak LC-MS/MS case in the biological sample of the mentioned control group, comparing the urine and dungs sample before and after the administrated Suoquan pills and Suoquan capsules, we found that the main substances detected in urine and dungs were prototype form and were not detected the phase Ⅱ metabolites, such as glucuronide metabolites, in rats after intragastric administration. Conclusion Nootkatone, puzzle ketone A and Young shoots flavin and other ingredients are detected in the urine of rats, which may be the active constituents in regulating the bladder and bladder function.