Widely Targeted Metabolomics Reveals Dynamic Secondary Metabolite Accumulation and Antioxidant Biomarkers Across Ripening Stages of Ziziphus jujuba cv. ‘Junzao’ Fruit

Fruit ripening is accompanied by extensive reprogramming of secondary metabolism, which determines the antioxidant value of medicine-food homologous fruits. Ziziphus jujuba Mill. cv. ‘Junzao’ (Junzao) is a high-quality cultivar rich in bioactive compounds, yet its stage-dependent metabolite accumulation and the corresponding antioxidant capacity remain poorly resolved. In this study, widely targeted metabolomics was combined with the quantification of total phenolic (TPC), total flavonoid (TFC), and total triterpenoid (TTC) contents and with 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS) radical-scavenging assays to profile Junzao fruits at five developmental stages. A total of 2388 secondary metabolites were identified, with flavonoids and terpenoids representing the major classes. TPC and TFC were highest at the immature YG (young-fruit) stage, whereas TTC peaked at the BS (white-ripe) stage; all three decreased during subsequent ripening, consistent with the stronger DPPH and ABTS radical-scavenging activities observed in early-stage fruits. Multivariate analyses revealed distinct metabolic profiles among developmental stages, and 2111 differentially accumulated metabolites (DAMs) were identified. K-means clustering resolved nine temporal accumulation patterns, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment indicated dynamic regulation of flavonoid biosynthesis, phenylpropanoid metabolism, and triterpenoid-related pathways. Spearman correlation analyses further identified ten metabolites, comprising six flavonoids, three triterpenes, and one phenolic acid, that were strongly associated with antioxidant capacity (|r| ≥ 0.5, p < 0.05), highlighting their potential as biomarkers for quality evaluation. Overall, immature Junzao fruits exhibited superior antioxidant capacity, supporting their promise as functional-food ingredients and providing a basis for stage-specific harvesting and utilization.