运用超高效液相色谱-生物化学检测在线联用分析方法筛选中药中的丁酰胆碱酯酶抑制剂

Screening and identifying butyrylcholinesterase inhibitors in traditional Chinese medicine by using on-line ultra-performance liquid chromatography coupled with biochemical detection

目的:建立超高效液相色谱(UPLC)-生物化学检测(BCD)在线联用技术快速筛选中药中的丁酰胆碱酯酯酶(BChE)抑制剂的方法。方法:运用UPLC-DAD-BCD方法快速筛选吴茱萸、钩藤、苦参、山豆根提取物中的BChE抑制剂。以莲心碱为阳性对照,考察UPLC-DAD-BCD方法用于BChE抑制剂筛选的可行性。运用超高效液相-线性离子阱/静电场轨道阱高分辨质谱(UHPLC-LTQ/Orbitrap MS)方法鉴定4种中药的70%甲醇提取物中的主要活性成分。结果:UPLC-DAD-BCD方法快速筛选出12个BChE抑制剂,其中吴茱萸4个、钩藤3个、苦参5个、山豆根2个,山豆根中的2个活性成分与苦参共有,通过UHPLC-LTQ/Orbitrap MS鉴定出其中的8个BChE抑制剂,分别为去氢吴茱萸碱、吴茱萸碱、吴茱萸次碱、1-甲基-2-[(6Z,9Z)-6,9-十五二烯基]-4(1H)-喹诺酮、去氢毛钩藤碱、毛钩藤碱、苦参碱和氧化苦参碱。结论:建立的UPLC-DAD-BCD在线分析方法灵敏度高、重现性好,能快速筛选中药中的BChE抑制剂,为中药中新的生物活性成分的快速筛选提供了一种高效、便捷的分析策略。

OBJECTIVE To establish an on-line ultra-performance liquid chromatography-biochemical detection(UPLC-BCD)method for screening butyrylcholinesterase(BChE)inhibitors in traditional Chinese medicinemethanol extracts.METHODS BChE inhibitors in methanol extracts of Fructus Evodiae,Uncaria Rhynchophylla,Radix Sophorae Flavescens and Radix Sophorae Tonkinensis were screened by the on-line method of UPLC-DAD-BCD.The feasibility was examined using liensinine as a positive reference compound.The major active ingredients in four herbs were identified by ultra-high-performance liquid chromatography linear ion trap/Orbitrap high resolution mass spectrometry(UHPLC-LTQ/Orbitrap MS).RESULTS Twelve BChE inhibitors were detected by on-line UPLC-DAD-BCD,including 4 inhibitors in Fructus Evodiae,3 inhibitors in UncariaRhynchophylla,5 inhibitors in Radix Sophorae Flavescens and 2 inhibitors in Radix Sophorae Tonkinensis.The two active components in Radix Sophorae Tonkinensis were also detected in Radix Sophorae Flavescens.And 8/12 BChE inhibitors,such as dehydroectonine,ectonine,ectoninine,1-methyl-2-[(6Z,9Z)-6,9-15 adienyl]-4(1H)-quinolone,dehydrodryclorhynine,dryclorhynine,matrine and oxymatrine,were tentatively identified by UPLC-LTQ/Orbitrap MS.CONCLUSION The established on-line UPLC-DAD-BChE method possesses excellent sensitivity and stability of quickly screening BChE inhibitors in herbal medicines.Thus a convenient and efficient analytical strategy has been formulated for rapid and targeted screening of new bioactive compounds in traditional Chinese medicine materials.