The radical scavenging activity of the U. tomentosa extracts was determined by the ORAC method as previously described [14 (link),29 (link)] using fluoresceine as the fluorescence probe. A Polarstar Galaxy plate reader (BMG Labtechnologies GmbH, Offenburg, Germany) with 485-P excitation and 520-P emission filters was used. Black 96-well untreated microplates (Nunc, Denmark) were used for fluorescence measurements in the Polarstar equipment controlled by Fluostar Galaxy software (v.4.11-0, BMG Labtechnologies GmbH, Offenburg, Germany). The reaction mixture placed in each well consisted of 200 µL of a mixture of fluorescein (70 nM, used as a fluorescence probe), AAPH (12 mM), and antioxidant: Either Trolox (1–8 µM) or U. tomentosa extract (at different concentrations). APPH and Trolox solutions were freshly prepared. Fluorescein was freshly diluted from a stock solution (1.17 mM) using 75 mM phosphate buffer (pH 7.4). The reaction was performed at 37 °C. The plate was automatically shaken before the first reading and afterwards fluorescence was measured every minute for 98 min. The curve of the blank (with no antioxidant) was used to normalize the fluorescence measurements and, from the fluorescence normalized curves, the area under the decay curve (AUC) was calculated as follows:
where f0 is the initial fluorescence reading at 0 min and fi is the fluorescence reading at time i. Finally, the net AUC that corresponds to a sample was calculated using the following formula:
The regression equation between the net AUC and the antioxidant concentration was therefore calculated. The ORAC value was in turn calculated by dividing the slope of the latter equation by the slope of the Trolox line attained for the same assay. Final ORAC values were expressed as mmol of Trolox equivalents (TE)/g of extract. Each reaction mixture was carried out in duplicate, and three independent runs were performed for each sample.
where f0 is the initial fluorescence reading at 0 min and fi is the fluorescence reading at time i. Finally, the net AUC that corresponds to a sample was calculated using the following formula:
The regression equation between the net AUC and the antioxidant concentration was therefore calculated. The ORAC value was in turn calculated by dividing the slope of the latter equation by the slope of the Trolox line attained for the same assay. Final ORAC values were expressed as mmol of Trolox equivalents (TE)/g of extract. Each reaction mixture was carried out in duplicate, and three independent runs were performed for each sample.
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