Detection of Antioxidant Active Components and Their Antioxidant Ability from Moutan Cortex by Ultra-High Performance Liquid Chromatography-Tandem Quadrupole Time-of-Flight Mass Spectrometry

In order to accurately detect the antioxidant active components and their antioxidant ability in Moutan Cortex samples with complex matrix, the title study was conducted, and the reaction mechanism of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical elimination process was speculated. Extraction was conducted twice on the sample by ultrasound with 70% (volume fraction, the same below) ethanol solution. After filtration, the filtrate was collected, concentrated, and centrifuged. The supernatant was diluted to prepare 200 g·L⁻¹ sample solution, and further diluted to prepare sample solution series with mass concentrations of 8, 40, 100, 200, 500, 1 000, 5 000, 10 000, 25 000 mg·L⁻¹. 300 μL of the prepared sample solution series mentioned above (sample control group without sample addition) and 600 μL of anhydrous ethanol, together with 600 μL of 50 mg·L⁻¹ DPPH solution were added. The mixed solution was vortexed for 1 min, reacted in dark for 30 min, and diluted 200 times with 70% ethanol solution. The solution obtained was passed through a 0.22 μm filtrate membrane, and the filtrate was analyzed by ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), to measure the peak areas P₀ and P of DPPH reference molecule ions before and after sample addition, and calculate the DPPH free radical scavenging rate using 100%×(P₀-P)/P₀. Various antioxidant active components were identified by combining their own spectrum library, reference substance spectra, and relevant literatures on Moutan Cortex. It was shown that linear relationship between values of the mass concentration of the sample and the DPPH free radical scavenging rate was kept in the range of 8－1 000 mg·L⁻¹. The proposed method was less affected by the interference of the sample matrix and color compared to the enzyme-linked immunosorbent assay method. The results of the DPPH free radical scavenging rate were consistent with those given by the enzyme-linked immunosorbent assay method. 18 antioxidant active components were significantly identified from Moutan Cortex, including 15 identifiable components (containing two pairs of isomers) and 3 unknown components. Among them, oxypaeoniflorin, paeonol, mudanpioside H, galloyl-paeoniflorin, methyl gallate, mudanpioside F, and 4-hydroxybenzoic acid were the main antioxidant active components in Moutan cortex.