This study investigated the protective effects of aged Pericarpium Citri Reticulatae ‘Chachi’ (PCR-C) on tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in HepG2 cells. According to HPLC analysis, PCR-C aged 10 years (PCR-C10) had the highest flavonoids content, especially polymethoxyflavones (PMFs), compared with the fresh peel of Citrus reticulata cv. ‘Chachiensis’ and PCR-C aged 1, 3, and 5 years. Then, flavonoids-rich PCR-C samples and non-flavonoids-rich PCR-C samples (NF) were prepared by extracting and purifying PCR-C of different aging periods, for further cell experiments. Pretreatment with flavonoids-rich PCR-C samples (particularly PCR-C10) considerably reversed t-BHP-induced oxidative damage in HepG2 cells by improving cell viability, increasing SOD activity and GSH levels and reducing the overproduction of ROS and MDA. Correlation analysis further indicated that the accumulation of PMFs, mainly 5,6,7,4′-tetramethoxyflavone and nobiletin, was the main reason that PCR-C10 maintained the redox balance in HepG2 cells. These findings provided direct evidence for the cellular antioxidant activity of aged PCR-C and a guide for PCR-C’s classification, authentication and rational use.

Pericarpium Citri Reticulatae ‘Chachi’ (PCR-C), rich in polymethoxyflavones (PMFs), has potential anticancer bioactivity and its quality will be improved during storage. However, the main factors influencing the PCR-C quality during its storage remain unclear. In this study, multivariate analysis was performed to investigate free and bound PMFs of PCR-C during storage. The anticancer effects of purified PCR-C flavonoid extracts (PCR-CF) and the important PMFs were evaluated using A549 cells. The results showed that PCR-C samples exhibited remarkable differences in free PMFs during storage, which fell into three clusters: Cluster 1 included fresh (fresh peel) and PCR-C01 (year 1); Cluster 2 consisted of PCR-C03 (year 3) and PCR-C05 (year 5); and PCR-C10 (year 10) was Cluster 3. 3,5,6,7,8,3′,4′-heptamethoxyflavone, tangeretin, and isosinensetin were identified as the most important PMFs distinguishing the various types of PCR-C according to its storage periods. Moreover, PCR-CF inhibited A549 cell proliferation and induced cell cycle arrest at G2/M phase, cell apoptosis, and ROS accumulation, and all anticancer indices had an upward tendency during storage. Additionally, tangeretin and 3,5,6,7,8,3′,4′-heptamethoxyflavone exhibited anticancer effects on A549 cells, whereas isosinensetin displayed no anticancer effect, indicating that tangeretin and 3,5,6,7,8,3′,4′-heptamethoxyflavone jointly contributed to anticancer activity of PCR-C during storage. PCR-CF and the most important PMFs killed cancer cells (A549 cells) but had no cytotoxicity to normal lung fibroblast cells (MRC-5 cells). Overall, the high quality of long-term stored PCR-C might be due to the anticancer effects of tangeretin and 3,5,6,7,8,3′,4′-heptamethoxyflavone.