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In this study, five flavonoid glycosides were extracted from defatted Camellia oleifera seeds by using a pressurized hot water extraction (PHWE) method. The independent experimental factors (temperature, time and pressure) influencing the PHWE efficiency of the flavonoid glycosides were optimized and quantified using an orthogonal array design and high-performance liquid chromatography – negative mode electron spray ionization/mass spectrometry (HPLC/(−)ESI–MS-MS), respectively. Based on the result of experimental design, the maximum extraction efficiency of flavonoid glycosides were obtained by selecting temperature at 140 °C, pressure at 600 psi, and time for 10 min. The recovery and overall yield of the flavonoid glycosides were greater than those in ultrasonic extraction (UE) (78.5% vs 68.4% and 18.8 mg/g vs 15.91 mg/g). In particularly, compounds 1 and 2, which are kaempferol derivatives, were higher 1.19–1.23 times than those of the compounds obtained through UE (6.63 mg/g vs 5.56 mg/g; 11.89 mg/g vs 9.66 mg/g, respectively). These two compounds have higher polarities and antioxidant capacities than the other three flavonoid glycosides. Thus, PHWE is an excellent alternative method for the extraction of flavonoid glycosides that are highly polar and potent antioxidants. [ABSTRACT FROM AUTHOR]