High-performance thin-layer chromatography – antibacterial assay first reveals bioactive clerodane diterpenes in giant goldenrod (Solidago gigantea Ait.)

Abstract

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 (IC50) 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.