Department of Physical Chemistry, Medical University of Lublin, Chodźki 4a, 20-093 Lublin, Poland.
Department of Inorganic Chemistry, Medical University of Lublin, Chodźki 4a, 20-093 Lublin, Poland.
Molecules. 2022 Jun 3;27(11):3606. doi: 10.3390/molecules27113606.
An important strategy for treating neurodegenerative disorders is to maintain the levels of acetylcholine in the synaptic cleft by blocking the cholinesterases. Searching for new effective compounds with inhibited acetylcholinesterase and butyrylcholinesterase activity is one of the most significant challenges of the modern scientific research. The aim of this study was the optimization of the condition for cholinesterase activity determination by high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) in terms of concentrations of enzymatic reaction mixture components, temperature of incubation, and incubation time. In vitro investigation of acetylcholinesterase and butyrylcholinesterase activity inhibition by some isoquinoline alkaloids and extracts obtained from the aerial part and roots of collected in May, July, and September. Acetylcholinesterase and butyrylcholinesterase activity inhibition of the extracts obtained from the plant had not been tested previously. The application of the HPLC method allowed eliminating absorption of interfering components, for example, alkaloids such as sanguinarine and berberine. The HPLC method was successfully applied for the evaluation of the acetylcholinesterase inhibitory activity in samples such as plant extracts, especially those containing colored components adsorbing at the same wavelength as the adsorption wavelength of 5-thio-2-nitro-benzoic acid, which is the product of the reaction between thiocholine (product of the hydrolysis of acetyl/butyrylthiocholine reaction) with Ellman's reagent. Moreover, liquid chromatography coupled with a triple quadrupole mass spectrometer (LC-QqQ-ESI-MS/MS) analysis allowed evaluating the identification of relevant bioactive compounds in the obtained plant extracts. The investigated alkaloids, especially sanguinarine and chelerythrine, and all the extracts, especially the extract obtained from the aerial part collected in May, exhibited very high cholinesterase activity inhibition. HPLC-DAD was also applied for the kinetics study of the most active alkaloids sanguinarine and chelerythrine. Our investigations demonstrated that these plant extracts can be recommended for further in vivo experiments to confirm their cholinesterase inhibition activity.
治疗神经退行性疾病的一个重要策略是通过抑制胆碱酯酶来维持突触间隙中的乙酰胆碱水平。寻找具有抑制乙酰胆碱酯酶和丁酰胆碱酯酶活性的新有效化合物是现代科学研究的最重大挑战之一。本研究的目的是优化高效液相色谱-二极管阵列检测（HPLC-DAD）条件，以确定酶反应混合物成分的浓度、孵育温度和孵育时间，从而确定胆碱酯酶活性。体外研究了五月、七月和九月采集的植物地上部分和根部分离得到的一些异喹啉生物碱和提取物对乙酰胆碱酯酶和丁酰胆碱酯酶活性的抑制作用。以前从未测试过从植物中获得的提取物对乙酰胆碱酯酶和丁酰胆碱酯酶活性的抑制作用。HPLC 方法的应用消除了吸收干扰成分的可能性，例如血根碱和小檗碱等生物碱。该 HPLC 方法成功应用于评估植物提取物等样品中的乙酰胆碱酯酶抑制活性，特别是那些含有在与 5-硫代-2-硝基苯甲酸（硫代胆碱与 Ellman 试剂反应的产物）的吸收波长相同的波长处吸附的有色成分的提取物。此外，液相色谱-三重四极杆质谱联用（LC-QqQ-ESI-MS/MS）分析允许评估获得的植物提取物中相关生物活性化合物的鉴定。研究的生物碱，特别是血根碱和白屈菜红碱，以及所有提取物，特别是五月采集的地上部分提取物，对胆碱酯酶表现出非常高的抑制活性。HPLC-DAD 还用于最活跃的生物碱血根碱和白屈菜红碱的动力学研究。我们的研究表明，这些植物提取物可推荐用于进一步的体内实验，以确认其对胆碱酯酶的抑制活性。
