Uncovering the antioxidant characteristics of black tea by coupling in vitro free radical scavenging assay with UHPLC-HRMS analysis

Black tea (BT) is rich in dietary antioxidants, but its antioxidant composition has not been fully understood. To identify the true antioxidants occurring in BT, we established an approach based on 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay coupled with ultra-high performance liquid chromatography-high resolution mass spectrometry (DPPIT -UHPLC-HRMS). The employment of HRMS enable us to detect trace antioxidants, resolve co-eluted antioxidants, and characterize chemical structures of unknown antioxidants. In total, 56 phenolic compounds were screened as potential antioxidants from 106 compounds identified in BT. Catechol and pyrogallol were revealed as the key substructures in enhancing the antioxidant abilities of phenolic compounds. During BT brewing, high temperature with extended time promote antioxidant leaching but may induce the degradation of esterified and glycosylated compounds such as theaflavin-3-gallate and rutin. In conclusion, this work identified the true antioxidant constituents of BT, their structural characteristics, and their dynamic changes under various brewing conditions.