Centre for Agricultural Research, ELKH, Plant Protection Institute, Herman O. Str. 15, Budapest 1022, Hungary.
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Semmelweis University, Üllői Str. 26, Budapest 1085, Hungary.
J Chromatogr A. 2022 Aug 16;1677:463308. doi: 10.1016/j.chroma.2022.463308. Epub 2022 Jul 9.
The present work introduces a high-performance thin-layer chromatography (HPTLC)-direct bioautography method using the Gram-positive plant pathogenic bacterium, Rhodococcus fascians. The screening and isolation procedure comprised of a non-targeted high-performance thin-layer chromatography-effect-directed analysis (HPTLC-EDA) against Bacillus subtilis, B. subtilis subsp. spizizenii, R. fascians, and Aliivibrio fischeri, a targeted HPTLC-mass spectrometry (MS), and bioassay-guided column chromatographic (preparative flash and semi-preparative HPLC) fractionation and purification. The developed new separation methods enabled the discovery of four bioactive cis-clerodane diterpenes, solidagoic acid H (1), solidagoic acid E (2), solidagoic acid I (3), and solidagoic acid F (4), in the n-hexane extract of giant goldenrod (Solidago gigantea Ait.) leaf for the first time. These compounds were identified by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. The initially used HPTLC method (chloroform - ethyl acetate - methanol 15:3:2, V/V/V) was changed (to n-hexane - isopropyl acetate - methanol - acetic acid 29:20:1:1, V/V/V/V) to achieve the separation of the closely related isomer pairs (1-2 and 3-4). Compounds 1 and 3 exhibited moderate antibacterial activity against the Gram-positive B. subtilis subsp. spizizenii and R. fascians bacterial strains in microdilution assays with half-maximal inhibitory concentration (IC) values in the range of 32.3-64.4 µg/mL. The mass spectrometric fragmentation of the isolated compounds was interpreted and their previously published NMR assignments lacking certain resonances were completed.
本工作介绍了一种使用革兰氏阳性植物病原菌 Rhodococcus fascians 的高效薄层色谱（HPTLC）直接生物自显影方法。筛选和分离程序包括针对枯草芽孢杆菌、枯草芽孢杆菌亚种、Rhodococcus fascians 和 Aliivibrio fischeri 的非靶向高效薄层色谱效应定向分析（HPTLC-EDA）、靶向高效薄层色谱-质谱（HPTLC-MS）和生物测定指导的柱色谱（制备闪蒸和半制备 HPLC）分离和纯化。开发的新分离方法首次发现了四种生物活性的顺式- clerodane 二萜，即 solidagoic acid H（1）、solidagoic acid E（2）、solidagoic acid I（3）和 solidagoic acid F（4），存在于巨型一枝黄花（Solidago gigantea Ait.）叶的正己烷提取物中。这些化合物通过 1D 和 2D 核磁共振（NMR）光谱鉴定。最初使用的 HPTLC 方法（氯仿-乙酸乙酯-甲醇 15:3:2，V/V/V）被改变（正己烷-乙酸异丙酯-甲醇-乙酸 29:20:1:1，V/V/V/V）以实现紧密相关的异构体对（1-2 和 3-4）的分离。化合物 1 和 3在微量稀释测定中对革兰氏阳性枯草芽孢杆菌亚种和 Rhodococcus fascians 细菌菌株表现出中等的抗菌活性，半最大抑制浓度（IC）值在 32.3-64.4 µg/mL 范围内。分离化合物的质谱碎裂被解释，并完成了它们以前发表的 NMR 分配中缺少某些共振的部分。
