Abstract: In this study, ultrasonic-assisted ethanol extraction was used to extract flavonoids from Diaphragma juglandis fructus, the dry wooden diaphragm inside walnuts, and AB-8 macroporous resin was used to separate and purify the crude flavonoids, yielding six fractions. Their antioxidant properties including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) cation radical, hydroxyl radical, superoxide anion radical and reactive oxygen species (ROS) scavenging capacity, and iron ion reducing power were determined. The composition and content of flavonoids in the six fractions were analyzed by ultra-high performance liquid chromatography-triple quadruple tandem mass spectrometry (UPLC-QQQ-MS/MS). The results showed that the ABTS cation radical, hydroxyl radical and ROS scavenging capacity of all six samples were significantly higher than those of vitamin C (VC). Quercetin was the most abundant flavonoid with the highest concentration of 2 426.9 ng/mL, and rutin and gallic acid were hardly detectable. In addition to the reported flavonoids naringin, rutin, isoquercetin, hyperoside, dihydroquercetin, catechin, quercetin and gallic acid (a member of the polyphenol family), quercetin, astragaloside and epicatechin gallate were also detected. The correlation analysis between flavonoid content and antioxidant capacity showed that naringin content was significantly negatively correlated with DPPH radical and ABTS cation radical scavenging capacity, while catechin content was positively correlated with ROS scavenging capacity. The results of validation experiment showed that the ABTS cation radical scavenging capacity of quercetin was significantly higher than that of VC and mixed samples. This study lays a foundation for the study of the antioxidant capacity and structure-activity relationship of quercetin, naringin and catechin.

Key words: Diaphragma juglandis fructus; flavonoids; separation and purification; antioxidation; ultra-high performance liquid chromatography-triple quadruple tandem mass spectrometry; compositional analysis