Department of Pharmacognosy with Medicinal Plant Unit, Medical University of Lublin, 1 Chodźki Str., 20-093 Lublin, Poland.
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey.
Phytomedicine. 2019 Feb 15;54:259-264. doi: 10.1016/j.phymed.2018.10.030. Epub 2018 Oct 26.
Medicinal plants are a proven source of drug-like small molecules with activity towards targets relevant for diseases of the central nervous system (CNS). Plant species of the Apiaceae family have to date yielded a number of neuroactive metabolites, such as coumarin derivatives with acetylcholinesterase inhibitory activity or anti-seizure activity.
To accelerate the discovery of neuroactive phytochemicals with potential as CNS drug leads, we sought to rapidly isolate furanocoumarins, primary constituents of the dichloromethane (DCM) extract of the fruits of Peucedanum alsaticum L. (Apiaceae), using high-performance counter-current chromatography (HPCCC) and to evaluate their neuroactivity using both in vitro and in vivo microscale bioassays based on cholinesterase ELISAs and zebrafish epilepsy models.
In this study the DCM extract was subjected to HPCCC for the efficient separation (60 min) and isolation of furanocoumarins. Isolated compounds were identified with TOF-ESI-MS and NMR techniques and examined as inhibitors of AChE and BChE using ELISA microtiter assays. Anti-seizure properties of the extract and of the isolated compounds were evaluated using a zebrafish epilepsy model based on the GABA antagonist pentylenetetrazol (PTZ), which induces increased locomotor activity and seizure-like behavior.
The solvent system, composed of n-heptane, ethyl acetate, methanol and water (3:1:3:1, v/v/v/v), enabled the isolation of 2.63 mg lucidafuranocoumarin A (purity 98%) and 8.82 mg bergamottin (purity 96%) from 1.6 g crude DCM extract. The crude extract, at a concentration of 100 µg/ml, exhibited a weak inhibitory activity against acetylcholinesterase (AChE) (9.63 ± 1.59%) and a moderate inhibitory activity against butyrylcholinestrase (BChE) (49.41 ± 2.19%). Lucidafuranocoumarin A (100 µg/ml) was inactive against AChE but showed moderate inhibition towards BChE (40.66 ± 1.25%). The DCM extract of P. alsaticum fruits (0.62-1.75 µg/ml) and bergamottin (2-10 µm) exhibited weak anti-seizure activity, while lucidafuranocoumarin A (10-16 µm) was found to significantly inhibit PTZ-induced seizures. The percentage of seizure inhibition for the isolated compounds, at their most bioactive concentration, was 26% for bergamottin and 69% for lucidafuranocoumarin A.
Our findings underscore the utility of HPCCC for the rapid isolation of rare coumarin derivatives, and the potential of microscale in vivo bioassays based on zebrafish disease models for the rapid assessment of neuroactivity of these drug-like natural products.
药用植物是具有活性的小分子药物的可靠来源，这些小分子药物针对中枢神经系统（CNS）相关靶点。迄今为止，伞形科植物已产生了许多神经活性代谢物，如具有乙酰胆碱酯酶抑制活性或抗惊厥活性的香豆素衍生物。
为了加速发现具有 CNS 药物先导潜力的神经活性植物化学物质，我们试图使用高效逆流色谱法（HPCCC）快速分离 Peucedanum alsaticum L.（伞形科）果实中二氯甲烷（DCM）提取物中的呋喃香豆素，这是主要成分，并使用基于乙酰胆碱酯酶 ELISA 和斑马鱼癫痫模型的体外和体内微量生物测定来评估其神经活性。
在这项研究中，DCM 提取物通过 HPCCC 进行高效分离（60 分钟）和呋喃香豆素的分离。使用 TOF-ESI-MS 和 NMR 技术鉴定分离得到的化合物，并使用 ELISA 微量滴定法测定它们对 AChE 和 BChE 的抑制作用。使用基于 GABA 拮抗剂戊四氮（PTZ）的斑马鱼癫痫模型评估提取物和分离化合物的抗惊厥特性，PTZ 可诱导运动活动增加和类似癫痫的行为。
溶剂系统由正庚烷、乙酸乙酯、甲醇和水（3:1:3:1，v/v/v/v）组成，可从 1.6g 粗 DCM 提取物中分离出 2.63mg 白芷素 A（纯度 98%）和 8.82mg 佛手柑内酯（纯度 96%）。粗提取物在 100µg/ml 浓度下对乙酰胆碱酯酶（AChE）表现出微弱的抑制活性（9.63±1.59%），对丁酰胆碱酯酶（BChE）表现出中等抑制活性（49.41±2.19%）。白芷素 A（100µg/ml）对 AChE 无活性，但对 BChE 表现出中等抑制作用（40.66±1.25%）。P. alsaticum 果实的 DCM 提取物（0.62-1.75µg/ml）和佛手柑内酯（2-10µm）表现出微弱的抗惊厥活性，而白芷素 A（10-16µm）则显著抑制了 PTZ 诱导的惊厥。分离化合物在最具生物活性浓度时的惊厥抑制率为 26%的佛手柑内酯和 69%的白芷素 A。
我们的研究结果强调了 HPCCC 用于快速分离稀有香豆素衍生物的实用性，以及基于斑马鱼疾病模型的微量体内生物测定在快速评估这些类药天然产物神经活性方面的潜力。
