Antioxidant Capacity of Umbrian LBB Extract

The antioxidant capacity of umbrian LBB extract was determined using the ORAC method as previously reported [40 (link)]. We have chosen ORAC method because it is a robust and reliable method. In fact, the ORAC assay, other common measures of antioxidant capacity include ferric ion reducing antioxidant power and trolox equivalence antioxidant capacity assays and therefore is considered to be a preferable method because of its biologic relevance [41 ]. A duplicate extraction was performed for each sample and used to evaluate the lipophilic (L-ORACFL) and hydrophilic ORACFL (H-ORACFL) values. Evaluations of the lipophilic and hydrophilic ORACFL in the LBBs samples were performed separately, and the total antioxidant capacity (TAC) was calculated by adding the L-ORACFL and H-ORACFL values. The ORACFL assays were carried out on a FLUOstar OPTIMA microplate fluorescence reader (BMG LABTECH, Offenburg, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The procedure was based on the method of Zulueta et al. (2009) with slight modifications. Briefly, 2,20-azobis (2-methylpropionamide) dihydrochloride (AAPH) was used as a peroxyl radical generator, trolox was used as a reference antioxidant standard, and fluorescein was used as a fluorescent probe. The data are expressed as micromoles of trolox equivalents (TE) per gram of sample (μmol TE/g).