Non-targeted metabolomics coupled with simulated processing strategy reveals temperature-driven flavonoid and organic...

Flavonoids and organic acids are the primary bioactive components contributing to the functional value of Typhae pollen (TP), and their content and quality are influenced by various processing conditions. To investigate the overall characteristics and dynamic changes of these components during processing, a non-targeted metabolomics strategy was employed to systematically characterize the compounds present before and after TP processing and to identify differential markers. A total of 114 compounds were preliminarily identified in processed TP (CTP). Based on PCA and PLS-DA analyses, 63 and 20 markers were screened in the negative and positive ion modes, respectively, to differentiate raw TP from CTP, using a VIP > 1 criterion. Among these, 18 differential markers were ultimately confirmed using significance thresholds of FC > 1.5 and p-value <0.05, in addition to comparisons with reference standards. During processing, 5-hydroxymethyl-2-furancarboxylic acid and 2-furoic acid were newly generated. The observed differences among samples primarily resulted from variations in flavonoid and organic acid content. Simulated processing revealed that high temperatures significantly degrade flavonoid glycosides through three mechanisms: hydrolysis of glycosidic bonds, rapid conversion of intermediates, and extensive structural disruption. Flavonoid glycosides underwent hydrolysis and deglycosylation, yielding the corresponding secondary glycosides or aglycones. Free sugar residues likely participated in Maillard or caramelization reactions, resulting in the formation of the two furanic organic acids. Processing TP to the P2-P3 levels most effectively promoted the generation of flavonoid aglycones (quercetin, kaempferol, and isorhamnetin) and the two furanic organic acids. At this level, the appearance characteristics of CTP satisfy both the traditional "processing" endpoint criteria and the standards outlined in the Chinese Pharmacopoeia. These compounds are recommended as chemical markers for objectively evaluating the TP processing procedure. By linking specific patterns of chemical transformation to measurable quality attributes, this study provides theoretical support for the development of TP processing standards and offers practical guidance for industrial production.