Chair of Food Science, Institute of Nutritional Science and Interdisciplinary Research Center, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany; Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6-10, 35032 Marburg, Germany; Pharmacognosy Department, Faculty of Pharmacy, Al-Azhar University, 11371 Cairo, Egypt.
Saint Catherine Protectorate, Nature Conservation Sector, Egyptian Environmental Affairs Agency, 64616 South Sinai, Egypt.
J Chromatogr A. 2019 Nov 8;1605:460371. doi: 10.1016/j.chroma.2019.460371. Epub 2019 Jul 15.
The genus Primula (Primulaceae) comprises species with high medicinal as well as ornamental values. Plants belonging to this genus are well recognized for their richness in bioactive constituents. The huge variety of secondary metabolites make their complete analysis impossible. In order to cope with this challenge, effect-directed analysis (EDA) via HPTLC coupled to structure elucidation techniques was applied on Primula species for the first time. As straightforward non-target bioanalytical technique, HPTLC-UV/Vis/FLD-EDA-ESI-HRMS hyphenates three different orthogonal dimensions, i.e. chromatography with spectrometric detection, biological/enzymatic assays and HRMS. The bioactive metabolites were determined in the middle polar extracts of two Primula species, P. veris (flower) and P. boveana (leaf). The bioactivity profiling comprised the antibacterial activity against Aliivibrio fischeri and Bacillus subtilis bacterial strains and acetyl-/butyrylcholinesterase (AChE/BChE) inhibition assays. The compounds were characterized and identified via their recorded spectral data (HRMS and H NMR). The results showed that linoleic and linolenic acids were the principle bioactive compounds present in the studied P. veris flower extract. In the P. boveana leaf extract, flavone, 2'-methoxy-, 2'-hydroxy- and 5,6,2',6'-tetramethoxyflavone (zapotin) were determined as active metabolites. The identification of zapotin, which was previously undescribed in the investigated plant, was considered as the strength of the straightforward non-target bioanalytical technique. Flavone turned out to be the highest potent metabolite, and at the same time, a multipotent compound referring to its various bioactivities discovered. An equivalency calculation of the HPTLC-AChE inhibition by flavone was performed with reference to the well-known inhibitor rivastigmine. As a result, the amount of flavone contained in 10.0 μg dry powder of P. boveana (corresponding to 0.1 μL extract) inhibited as strong as 4.5 μg rivastigmine. In other words, the flavone contained in P. boveana leaf extract powder turned out to be half as strong as the well-known AChE inhibitor rivastigmine.
报春花属（报春花科）包含具有高药用和观赏价值的物种。该属植物以富含生物活性成分而闻名。大量的次生代谢产物使得对其进行完全分析变得不可能。为了应对这一挑战，首次将基于高效薄层色谱（HPTLC）的定向生物活性分析（EDA）技术应用于报春花属植物。作为一种直接的非靶向生物分析技术，HPTLC-UV/Vis/FLD-EDA-ESI-HRMS 串联了三个不同的正交维度，即色谱与光谱检测、生物/酶测定和 HRMS。从中等极性提取物中测定了两种报春花属植物，报春花（花）和报春花（叶）的生物活性代谢物。生物活性分析包括对发光弧菌和枯草芽孢杆菌的抗菌活性以及乙酰胆碱酯酶/丁酰胆碱酯酶（AChE/BChE）抑制测定。通过记录的光谱数据（HRMS 和 1H NMR）对化合物进行了表征和鉴定。结果表明，亚油酸和亚麻酸是研究中报春花花提取物中主要的生物活性化合物。在报春花叶提取物中，鉴定出黄酮、2'-甲氧基-、2'-羟基-和 5,6,2',6'-四甲氧基黄酮（扎波廷）为活性代谢物。扎波廷在研究植物中以前没有被描述，它的鉴定被认为是这种直接非靶向生物分析技术的优势。黄酮是最有效的代谢物，同时也是一种具有多种生物活性的多功能化合物。根据众所周知的抑制剂利斯的明，对黄酮的 HPTLC-AChE 抑制作用进行了等效计算。结果表明，10.0μg 干燥的报春花（相当于 0.1μL 提取物）中的黄酮含量与 4.5μg 利斯的明的抑制作用相当。换句话说，报春花叶提取物粉末中的黄酮含量与著名的 AChE 抑制剂利斯的明的抑制作用相当。
