Combinatorial phytochemical investigation: Systematic anti-oxidant constituent identification and chemical markers discovery in immature <i>Citrus</i> oils via temperature-stratified distillation metabolomics

<i>Citrus reticulata</i> ‘Chachi’ <i>Fructus Citri Immaturus</i> (CRC-FCI), a medicinal <i>Citrus</i> cultivar indigenous to Xinhui, China, contains bioactive essential oils (EOs) with phytochemical profiles that remain incompletely characterized. In this study, CRC-FCI-EOs were analyzed through temperature-stratified distillation (95-260°C) combined with GC-MS/FID analysis, leading to the identification of 95 constituents. Antioxidant assays (DPPH, ABTS, and FRAP) revealed temperature-independent antioxidant capacity in EO fractions collected above 140°C (140-260°C range). Multivariate analysis (PCA/OPLS-DA) identified eight differential metabolites, with γ-terpinene, β-caryophyllene, and thymol emerging as core antioxidant constituents exhibiting dose-dependent efficacy. Cellular studies demonstrated that EO fractions significantly reduced lipid peroxidation products-malondialdehyde (MDA) by 37.5-54.4% and reactive oxygen species (ROS) levels by 14.4-37.7%-while enhancing antioxidant enzyme activities in oxidative stress models: glutathione peroxidase (GPx, 1.5-2.5-fold), superoxide dismutase (SOD, 0.95-1.9-fold), and catalase (CAT, 1.2-2.1-fold). Notably, five chemical markers, D-limonene, γ-terpinene, methyl 2-(methylamino) benzoate, terpinolene, and <i>p</i>-cymene, were established for CRC-FCI differentiation. These findings provide critical phytochemical references for natural antioxidant discovery and quality control of CRC-FCI.