Department of Pharmacognosy, Medical University of Gdansk, al. gen. J. Hallera 107, 80-416, Gdansk, Poland.
Phytochem Anal. 2014 Jan-Feb;25(1):29-35. doi: 10.1002/pca.2455. Epub 2013 Jul 9.
Hyoscyamine and scopolamine, anti-cholinergic agents widely used in medicine, are typically obtained from plants grown under natural conditions. Since field cultivation entails certain difficulties (changeable weather, pests, etc.), attempts have been made to develop a plant in vitro culture system as an alternative source for the production of these compounds. During experiments to locate the limiting steps in the biotechnological procedure, it is important to monitor not only the levels of the final products but also the changes in the concentration of their precursors.
To develop a HPTLC method for the separation and quantitation of the main tropane alkaloids hyoscyamine and scopolamine, their respective direct precursors littorine and anisodamine, and cuscohygrine, a product of a parallel biosynthetic pathway that shares a common precursor (N-methyl-∆(1) -pyrrolium cation) with tropane alkaloids.
Using alkaloid extracts from Atropa baetica hairy roots, different TLC chromatographic systems and developing procedures were investigated.
Full separation of all compounds was obtained on HPTLC Si60 F254 plates preconditioned with mobile phase vapours (chloroform:methanol:acetone:25% ammonia ratios of 75:15:10:1.8, v/v/v/v). The chromatograms were developed twice (at distances of 4.0 and 3.0 cm) in a Camag twin trough chamber and visualised with Dragendorff's reagent. Densitometric detection (λ = 190 and 520 nm) was used for quantitative analyses of the different plant samples.
This method can be recommended for quantitation of hyoscyamine, scopolamine, anisodamine, littorine and cuscohygrine in different plant material (field grown vs. in vitro cultures).
莨菪碱和东莨菪碱是广泛应用于医学的抗胆碱能药物，通常从自然条件下生长的植物中提取。由于田间种植存在一定的困难（如天气变化、病虫害等），因此人们试图开发一种植物的体外培养系统作为这些化合物的替代来源。在寻找生物技术过程中的限制步骤的实验中，重要的是不仅要监测最终产物的水平，还要监测其前体浓度的变化。
开发一种 HPTLC 方法，用于分离和定量主要托烷生物碱莨菪碱和东莨菪碱、它们各自的直接前体天仙子胺和山莨菪碱，以及可卡因，它是一种平行生物合成途径的产物，与托烷生物碱共享一个共同的前体（N-甲基-∆(1)-吡咯阳离子）。
使用 Atropa baetica 发根的生物碱提取物，研究了不同的 TLC 色谱系统和展开程序。
在经过流动相蒸气预处理的 HPTLC Si60 F254 板上（氯仿：甲醇：丙酮：25%氨的比例为 75:15:10:1.8，v/v/v/v），所有化合物都得到了完全分离。在 Camag 双槽室中，将色谱图展开两次（距离分别为 4.0 和 3.0 cm），并用 Dragendorff 试剂显色。采用分光光度检测（λ=190 和 520nm）对不同植物样品进行定量分析。
该方法可推荐用于不同植物材料（田间种植与体外培养）中莨菪碱、东莨菪碱、山莨菪碱、天仙子胺和可卡因的定量分析。
