背景:苦参(豆科),亚洲一种无处不在的植物,含有广泛的药理活性化合物,如类黄酮,具有潜在的抗阿尔茨海默病(抗AD)作用。
目的:本研究的目的是开发一种四元筛选方法,隔离,提取优化,并对苦参乙酰胆碱酯酶(AChE)抑制化合物进行活性评价,实现中药活性物质的高通量筛选,为抗AD药物的开发提供实验数据。
方法:以AChE为靶分子,应用亲和超滤和液相色谱-质谱联用技术筛选苦参中潜在的酶抑制剂。首次采用正交阵列实验结合多目标非支配排序遗传算法III优化提取活性物质的工艺。进行酶抑制动力学和分子对接研究以验证活性化合物的潜在抗AD作用。
结果:确定了5种AChE抑制化合物:kushenolI,kurarinone,苦参酮G,异okurinone,和苦参醇E。它们以72.88%的纯度成功分离,98.55%,96.86%,96.74%,和95.84%,分别,使用正己烷/乙酸乙酯/甲醇/水(4.0/5.0/4.0/5.0,v/v/v/v),正己烷/乙酸乙酯/甲醇/水(5.0/5.0/6.0/4.0,v/v/v),和正己烷/乙酸乙酯/甲醇/水(4.9/5.1/5.7/4.3,v/v/v)流动相系统。酶抑制动力学表明,苦参醇E的抑制作用最好。
结论:本研究阐明了5种活性AChE抑制剂对苦参的作用机制,为抗AD等治疗药物的筛选和开发提供了理论依据。
BACKGROUND: Sophora flavescens Aiton (Fabaceae), a ubiquitous plant species in Asia, contains a wide range of pharmacologically active compounds, such as flavonoids, with potential anti-Alzheimer\'s disease (anti-AD) effects.
OBJECTIVE: The objective of the study is to develop a quaternity method for the screening, isolation, extraction optimization, and activity evaluation of acetylcholinesterase (AChE)-inhibiting compounds from S. flavescens to realize high-throughput screening of active substances in traditional Chinese medicine and to provide experimental data for the development of anti-AD drugs.
METHODS: With AChE as the target molecule, affinity ultrafiltration and liquid chromatography-mass spectrometry were applied to screen for potential inhibitors of the enzyme in S. flavescens. Orthogonal array experiments combined with the multi-objective Non-Dominated Sorting Genetic Algorithm III was used for the first time to optimize the process for extracting the active substances. Enzyme inhibition kinetics and molecular docking studies were performed to verify the potential anti-AD effects of the active compounds.
RESULTS: Five AChE-inhibiting compounds were identified: kushenol I, kurarinone, sophoraflavanone G, isokurarinone, and kushenol E. These were successfully separated at purities of 72.88%, 98.55%, 96.86%, 96.74%, and 95.84%, respectively, using the n-hexane/ethyl acetate/methanol/water (4.0/5.0/4.0/5.0, v/v/v/v), n-hexane/ethyl acetate/methanol/water (5.0/5.0/6.0/4.0, v/v/v/v), and n-hexane/ethyl acetate/methanol/water (4.9/5.1/5.7/4.3, v/v/v/v) mobile phase systems. Enzyme inhibition kinetics revealed that kushenol E had the best inhibitory effect.
CONCLUSIONS: This study elucidates the mechanism of action of five active AChE inhibitors in S. flavescens and provides a theoretical basis for the screening and development of anti-AD and other therapeutic drugs.