Anti-inflammatory markers of Ocotea (Lauraceae) uncovered through concatenated UPLC/MS-NMR metabolomics – dual inhibition of COX and LOX pathways

The Lauraceae family, particularly the genus Ocotea, is recognized for its wide chemical diversity and medicinal properties, such as anti-inflammatory. Inflammation, driven by pathways such as cyclooxygenase (COX) and lipoxygenase (LOX), plays a central role in several human pathologies. These pathways are essential for the production of key pro-inflammatory mediators, such as prostaglandin E2 (PGE2) and leukotriene B4 (LTB4). This study aimed to prospect dual COX/LOX inhibitory biomarkers in Ocotea species using a concatenated ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC/MS) - nuclear magnetic resonance (NMR) metabolomics approach and machine learning (ML) prediction models. Sixteen Ocotea species were analysed, with PGE2 and LTB4 levels determined by using an ex-vivo anti-inflammatory assay. The metabolomic data were subjected to block scaling and concatenated, with UPLC/MS and ¹H-NMR data analyzed as distinct blocks. Both unsupervised and supervised multivariate statistical methods were performed. Additional statistical total correlation spectroscopy (STOCSY) was performed to identify and correlate chemical shifts in the NMR data. In addition, gas-phase fragmentation reactions were proposed to increase metabolite confidence annotation in the UPLC/MS data. ML models were integrated to predict novel promissory extracts based on the discriminant bioactive markers of the metabolomics data. Nine Ocotea species showed dual inhibition, annotating two aporphine and two benzylisoquinoline alkaloids, one sesquiterpenoid and one glycosylated flavonoid as key potential anti-inflammatory compounds. This metabolomics approach also reinforces the anti-inflammatory potential of Ocotea species as a source for the discovery of novel anti-inflammatory agents with innovative mechanisms of action through dual inhibition of COX and LOX pathways. Keywords: Natural Products, Biochemometrics, Chemical profiling, Spectroscopy and Spectrometric Techniques, Inflammation.