Optimization of flavonoid extraction from Tarocco orange ( Citrus sinensis [L.] Osbeck) peel using supercritical CO 2 followed by ethanol: chemical composition, antioxidant, and antibacterial activities

Abstract BACKGROUND Blood orange peels represent an underutilized source of high‐value flavonoids with broad bioactivities. Traditional single‐extraction techniques for citrus flavonoids suffer from low efficiency due to polarity limitations, significantly restricting their industrial‐scale development and application. As a result, there is an urgent need to develop green and efficient extraction processes to improve both the coverage and yield of citrus flavonoids. RESULTS This study developed a two‐step combined extraction process to obtain flavonoids from blood orange peels: supercritical CO 2 extraction (SCE) followed by residue ethanol extraction (REE). Response surface methodology with Box–Behnken design was employed to optimize key process parameters for maximizing extraction efficiency. Compared to traditional solvent extraction, this process exhibited the following advantages: (1) qualitative analysis by ultra‐performance liquid chromatography–quadrupole–Orbitrap high‐resolution mass spectrometry identified seven additional flavonoids in the two‐step extract, revealing that SCE was primarily composed of low‐polarity flavonoids, and REE was primarily composed of high‐polarity flavonoids; (2) quantitative analysis by HPLC demonstrated that the two‐step extraction significantly enhanced flavonoid content, with total flavonoids increasing by 11.75% and polymethoxyflavones rising by 19.55%; (3) antioxidant and antimicrobial analyses revealed the potent antioxidant capacity of REE extracts and the superior antimicrobial activity of SCE extracts. CONCLUSION This study innovatively developed a two‐step combined extraction process for efficient stepwise enrichment of flavonoids with varying polarities from blood orange peel, significantly increasing the content of total flavonoids and polymethoxyflavones. For the first time, it elucidated the polarity complementarity between low‐polarity SCE and high‐polarity REE extracts, providing a novel strategy for polarity‐targeted flavonoid utilization. © 2025 Society of Chemical Industry.