Chemical and biological characterization of naturally aged GCP by using a rigorous sample collection protocol

Naturally aged GCP (peel of Citrus reticulata ‘ChaChi’) is a traditional medicine and food ingredient in China. Extensive studies have explored how the aging process affects the chemical and biological properties. In the present study, we used a rigorous sample collection protocol in which GCP were annually collected from a single citrus orchard and aged for 1 to 6 years in a sample warehouse for natural aging. A global chemical investigation was conducted using GC–MS and UHPLC-Orbitrap-MS, identifying 37 volatile and 112 non-volatile chemicals. Volatile terpenes and flavonoid glycosides metabolized into other constituents during aging via oxidation and hydrolysis. The contents of 55 major targets and pseudotargeted metabolomics analysis revealed distinct variation trends in polymethoxyflavones (PMFs) and limonoids. Antioxidant assays of volatile and non-volatile extracts indicated that four-year GCP samples exhibit the best antioxidant activity. The accumulation of terpenoid alcohols and sesquiterpenes, but not terpenes, presented significant positive correlations with antioxidant activity. For non-volatile constituents, PMFs and limonoids displayed significant positive correlations with antioxidant activity. RAW 264.7 cell test showed that all samples demonstrated moderate anti-inflammatory effects, and total PMFs correlated positively with anti-inflammatory activity. This study enhances understanding of the aging mechanism of GCP and establishes a rationale for its practical application.