Online RP-HPLC-DPPH screening method for detection of radical-scavenging phytochemicals from flowers of Acacia confusa

Acacia confusa is traditionally used as a medicinal plant in Taiwan. In this study, phytochemicals and antioxidant activities of extracts from flowers of A. confusa were investigated for the first time. In addition, a rapid screening method, online RP-HPLC-DPPH system, for individual antioxidants in complex matrices was developed. Accordingly, six antioxidants including gallic acid (1), myricetin 3-rhamnoside (2), quercetin 3-rhamnoside (3), kaempferol 3-rhamnoside (4), europetin 3-rhamnoside (5), and rhamnetin 3-rhamnoside (6) were detected using the developed screening method. Of these, compounds 2, 3, and 5 were found to be major bioactive phytochemicals, and their contents were determined as 11.3, 6.7, and 8.7 mg/g of crude extract, respectively. By comparison with quercetin, a well-known antioxidant, these compounds had the order of compound 2 > compound 5 > quercetin > compound 3 for DPPH radical-scavenging activity. Their IC₅₀ values were 3.0, 3.2, 4.5, and 7.4 μM, respectively. Moreover, the same order was observed for superoxide radical-scavenging activity, and their IC₅₀ values were 2.6, 2.7, 4.3, and 5.3 μM, respectively. However, for lipid peroxidation, quercetin, an aglycon, showed the best inhibitory activity. The IC₅₀ values of quercetin, compound 2, compound 5, and compound 3 were 46.7, 88.5, 90.7, and 124.6 μM, respectively. These results indicated that a rhamnoside at the C3 position of flavonoids had a negative effect on radical-scavenging activity and antilipid peroxidation. In contrast, the number of hydroxyl groups on the B-ring exhibited a positive relationship with their inhibitory activities.