Abstract
A practical TLC-direct bioassay method was established for screening and evaluating antioxidant activity. The method was based on series of reactions. Irradiation of linoleic acid with UV 254 nm produced lipid peroxides. Upon heating, the peroxides were converted into malondialdehyde, which combined with thiobarbituric acid to a red pigment with a maximum absorption at 535 nm. Following a TLC separation, substance with anti-lipid peroxidation activity showed white spots/zones against red background on the TLC plate. The proposed method was established by the dot blot test, and validated with respect to selectivity, sensitivity, linearity, precision, and recovery after optimizing key experimental parameters. In an exemplary screening for antioxidant constituents from Perilla fruits, two active zones were discovered and tentatively characterized as salviaflaside and rosmarinic acid by TLC-interface-ESI-MS, respectively. Further verification by HPTLC-bioassay-guided-isolation was carried out and in addition to salviaflaside and rosmarinic acid, also luteolin 5-O-β-d-glucopyranoside and 3′-dehydroxyl-rosmarinic acid-3-O-β-D-glucoside were discovered to show prominent anti-lipid peroxidation activity.
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