Discovery, Isolation, and Characterization of 23-SpirocholestaneDerivatives as Novel Plant Root Growth Inhibitors from Ypsilandra thibetica

In the present study, we first discovered that the 70% ethanol extract of Ypsilandra thibetica significantly inhibited <i>Arabidopsis</i> root growth. Through a bioactivity-guided strategy, 18 rare 23-spirocholestane derivatives (<b>1</b>–<b>18</b>), including 12 new ones (<b>1</b>–<b>12</b>), featuring a dioxane ring between C-24/C-23 of the aglycone moiety and C-1<i>′</i>/C-2<i>′</i> of the d-fucose ligand, were obtained from the strongest active elution fraction. Their structures were elucidated via a comprehensive method of MS and NMR spectroscopy, single-crystal X-ray diffraction, and chemical methods. Evaluation of their inhibitory effects of all isolates on <i>Arabidopsis</i> root growth revealed that <b>5</b>/<b>6</b>, <b>3</b>/<b>4</b>, <b>1</b>, and <b>2</b> exhibited pronounced activity, with EC₅₀ values of 6.89, 9.13, 18.01, and 26.83 μM, respectively. Then, a preliminary structure–activity relationship (SAR) analysis of these compounds was conducted. Furthermore, transcriptome data indicated that <i>Arabidopsis</i> responded to the stress caused by these bioactive compounds through the modulation of ethylene and auxin signaling pathways and the activation of various detoxification-related genes. Finally, the herbicidal evaluation showed that <b>3</b>/<b>4</b> and <b>5</b>/<b>6</b> significantly inhibited the root growth of Echinochloa crusgalli, while <b>3</b>/<b>4</b> remarkably suppressed the root growth of Lolium multiflorum. These findings highlight that 23-spirocholestane derivatives represent promising candidates for the development of new bioherbicides.