Untargeted LC–MS Metabolomics-Driven Discovery of Unique Peptides Cyanogristatins A–E from Sponge-Associated <i>Actinoalloteichus cyanogriseus</i> LHW52806 and Anti-Inflammatory Activity Assessment

This study established LC-MS-based metabolomics integrated with multivariate statistical analysis (UMMMS) to identify differential metabolites in <i>Actinoalloteichus cyanogriseus</i> under solid versus liquid culture conditions. Using this approach, 48 molecules exhibiting significant abundance differences between solid and liquid media were rapidly screened out. Among these, 8 were preliminarily identified as peptides potentially containing the unique β-Me-Leu residue by targeted MS/MS analysis. Guided by these screening results, 5 previously undiscovered peptides, designated as cyanogristatins A-E (<b>1</b>-<b>5</b>), were isolated. Their structures were elucidated using 1D/2D NMR spectroscopy, HR-MS/MS, and the advanced Marfey's method. Furthermore, <b>5</b> effectively reduced the number of migrating inflammatory cell aggregates in a zebrafish model of inflammatory bowel disease at a concentration of 20 μM. In subsequent experiments using an in vitro organoid model, <b>5</b> did not exert direct epithelial protective effects; however, ELISA further revealed that it decreased the level of production of TNF-α in intestinal macrophages. This finding suggests that the anti-inflammatory effect of <b>5</b> is most likely mediated by the regulation of the cytokine TNF-α. In summary, this study demonstrates the potential of mass spectrometry technology for the comprehensive analysis of natural products, facilitating the rapid discovery of novel compounds and enhancing the exploration of microbial secondary metabolites.