In vitro peroxynitrite scavenging activity of 6-hydroxykynurenic acid and other flavonoids from Gingko biloba yellow leaves

As part of our research on phytochemicals that exert protective effects against diseases related to reactive nitrogen species, we have evaluated the scavenging activity of the yellow leaves of Ginkgo biloba on ONOO^-. The methanol extract and ethyl acetate fraction obtained from yellow leaves of G. biloba evidenced a marked scavenging activity on authentic ONOO^-. Repeated column chromatography of the active ethyl acetate soluble fraction on silica gel, Sephadex LH-20, and RP-18, resulted in the purification of 15 known compounds, including sciadopitysin (1), ginkgolide B (2), bilobalide (3), isoginkgetin (4), kaempferol (5), luteolin (6), protocatechuic acid (7), bilobetin (8), amentoflavone (9), β-sitosterol glucopyranoside (10), kaempferol 3-O-rhamnopyranoside (11), kaempferol 3-O-glucopyranoside (12), kaempferol 3-O-[6‴-O-p-coumaroyl-β-D-glucopyranosyl(1→2)- α-L-rhamnopyranoside] (13), kaempferol 3-O-rutinoside (14), and 6-hydroxykynurenic acid (15). Among the compounds isolated, flavonoids (5, 6 and 11-14), protocatechuic acid (7), and 6-hydroxykynurenic acid (15) all exhibited marked scavenging activities on authentic ONOO^-. The IC₅₀ values of 5-7, 11-14 and 15 were as follows: 2.86 ± 0.70, 2.30 ± 0.04, 2.85 ± 0.10, 5.60 ± 0.47, 4.16 ± 1.65, 2.47 ± 0.15, 3.02 ± 0.48, and 6.24 ± 0.27 μM, respectively. DL-Penicillamine (IC₅₀ = 4.98 ± 0.27 μM) was utilized as a positive control. However, the other compounds (1-4, 8-10) exerted no effects against ONOO^-.