TPC, DPPH, ABTS and ORAC assays were selected for the characterisation of SBP extracts. Detailed description of these methods is provided elsewhere [27 (link)]. Briefly, for TPC assay extract solutions were mixed with Folin–Ciocalteau reagent and 7% Na2CO3 in a 96-well microplate. The absorbance was measured at 765 nm after 30 min in a FLUOstar Omega Reader (BMG Labtech, Offenburg, Germany). TPC was expressed in mg of GAE/g dry extract weight (DWE) and DWP.
For ABTS•+ decolourisation 6 µL of sample were added to 294 µL of ABTS•+ working solution, while for DPPH• scavenging 8 μL of sample were mixed with 292 µL of DPPH• methanolic solution. The absorbance was measured in 96-well microplates using a FLUOstar Omega Reader (BMG Labtech, Ortenberg, Germany) during 30 min at 734 nm and 60 min at 515 nm for ABTS•+ and DPPH•, respectively. Trolox was used as a standard, antioxidant capacity of the extracts was determined from the calibration curves and the results were expressed as µM TE/g DWE and DWP. Each analysis was carried out in six replicates.
For ORAC assay 25 µL of sample and 150 µL (14 μM) fluorescein solutions were placed into the wells of a black 96-well microplate. Then the mixture was preincubated in a FLUOstar Omega Reader for 15 min at 37 °C and 25 µL of AAPH (240 mM) were pipetted into each well. The fluorescence was recorded every cycle (in total, 120 cycles) using 485 excitation and 530 emission fluorescence filters. Antioxidant curves (fluorescence versus time) were first normalized and from the normalized curves the net area under the fluorescein decay curve (AUC) was measured. The results were expressed in µM TE/g DWE and DWP.
For ABTS•+ decolourisation 6 µL of sample were added to 294 µL of ABTS•+ working solution, while for DPPH• scavenging 8 μL of sample were mixed with 292 µL of DPPH• methanolic solution. The absorbance was measured in 96-well microplates using a FLUOstar Omega Reader (BMG Labtech, Ortenberg, Germany) during 30 min at 734 nm and 60 min at 515 nm for ABTS•+ and DPPH•, respectively. Trolox was used as a standard, antioxidant capacity of the extracts was determined from the calibration curves and the results were expressed as µM TE/g DWE and DWP. Each analysis was carried out in six replicates.
For ORAC assay 25 µL of sample and 150 µL (14 μM) fluorescein solutions were placed into the wells of a black 96-well microplate. Then the mixture was preincubated in a FLUOstar Omega Reader for 15 min at 37 °C and 25 µL of AAPH (240 mM) were pipetted into each well. The fluorescence was recorded every cycle (in total, 120 cycles) using 485 excitation and 530 emission fluorescence filters. Antioxidant curves (fluorescence versus time) were first normalized and from the normalized curves the net area under the fluorescein decay curve (AUC) was measured. The results were expressed in µM TE/g DWE and DWP.
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