Antioxidant activity in extracts of marine organisms screened by microtiter plate assay

A novel microtiter plate assay was developed to determine the total peroxyl radical-trapping activity of antioxidants extracted from marine organisms by measuring the inhibition rate of dye-substrate oxidation. The 96-well microtiter plate format afforded multisample throughput.Dihydrorhodamine-123 (DHR), dihydrofluorescein (DHR), and dichlorodihydrofluorescein (DCDHF) were used as reduced substrates for oxidation by peroxyl radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH).The antioxidant inhibition rates for uric acid and trolox were measured on addition of antioxidant reagents to the aqueous portion of the reaction mixture. Other comparative data were obtained by adjusting concentrations of the reduced substrates or concentrations of the AAPH peroxyl initiator.Data were prepared for graphic presentation, and reaction rates were determined by linear regression using Kaleida-Graph software (Synergy Software, Reading, Pa.).The inaugural test examined 166 methanolic extracts obtained from a selection of coral reef organisms and commensal microbes cultured on marine agar. Extracts included the marine sponges Paramyxilla n.sp. and Lissodendoryx isodicryalis; and the halophilic algae, Dampier Salt #1A.
To determine a simple, robust, and reliable method for screening antioxidant activity in extracts of marine organisms using microtiter plate technology to perform concurrent multi-sample analyses.To demonstrate a chromatographic strategy for examining the antioxidant composition of active extracts for novel lead selection, based on the principle of electrochemical detection.To design and test suitable procedures for shipboard analysis of freshly prepared extracts obtained from marine organisms.