Application of UPLC-MS/MS-Based Widely Targeted Metabolomics Reveals Metabolic Reprogramming During Post-Harvest...

Ehretia macrophylla fruit is a traditionally used but underutilized functional resource that is noted for its diverse phytochemical composition, which includes flavonoids and polyphenols. However, the systematic metabolic transformations that occur in the fruit during post-harvest storage-particularly those associated with its traditional processing into dark fruit tea-remain largely unknown, limiting efforts to optimize quality and processing strategies. In the study, to address this gap, we employed a widely targeted metabolomics approach based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to dynamically profile the fruit metabolome across four storage stages (T0, T4, T8, T12). A total of 1101 metabolites were putatively annotated, and day 8 (T8) emerged as a potential pronounced metabolic inflection point. From T8 onward, 30-40% of metabolites showed differential accumulation, characterized by a gradual rise in nutrition-relevant lipids, amino acids, and vitamins. In contrast, key antioxidant-related metabolites, including phenolics and flavonoids, increased transiently before T8 but declined sharply thereafter. These coordinated shifts correlated with an increased representation of lipid- and alkaloid-related metabolic pathways, whereas flavonoid-associated features showed a relative decline. Collectively, these findings provide insights into the biochemical processes potentially associated with fruit blackening and the marked decline in antioxidant capacity during traditional processing. Our study provides the first metabolic blueprint connecting traditional processing practices with phased metabolic remodeling, offering a scientific foundation for quality assessment and the development of informed post-harvest strategies for processing E. macrophylla fruit.