Antioxidant principles of Nelumbo nucifera stamens

In our ongoing study to identity antioxidants from natural sources, the antioxidant activity of Nelumbo nucifera stamens was evaluated for their potential to scavenge stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, inhibit total reactive oxygen species (ROS) generation, in kidney homogenates using 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA), and scavenge authentic peroxynitrites (ONOO^-). A methanol (MeOH) extract of the stamens of N. nucifera showed strong antioxidant activity in the ONOO^- system, and marginal activity in the DPPH and total ROS systems, so were therefore fractionated with several organic solvents, such as dichloromethane (CH₂Cl₂), ethyl acetate (EtOAc) and n-butanol (n-BuOH). The EtOAc soluble fraction, which exhibited strong antioxidant activity in all the model systems tested, was further purified by repeated silica gel and Sephadex LH-20 column chromatographies. Seven known flavonoids [kaempferol (1), kaempferol 3-O-β-D-glucuronopyranosyl methylester (2), kaempferol 3-O-β-D-glucopyranoside (3), kaempferol 3-O-β-D-galactopyranoside (4), myricetin 3′,5′-dimethylether 3- O-β-D-glucopyranoside (5), kaempferol 3-O-α-L-rhamnopyranosyl- (1→6)-β-D-glucopyranoside (6) and kaempferol 3-O-β-D- glucuronopyranoside (7)], along with β-sitosterol glucopyranoside (8), were isolated. Compound 1 possessed good activities in all the model systems tested. Compounds 2 and 7 showed scavenging activities in the DPPH and ONOO^- tests, while compounds 3 and 4 were only active in the ONOO^- test. Conversely, compound 8 showed no activities in any of the model systems tested.