The SOAC determination was done following previous procedures [21 (link),39 (link),40 (link)] with slight modifications. Briefly, 0.3 g of frozen flavedo tissue was extracted in 6 mL of cooled ethanol:chloroform:water (50:50:1, v:v:v) using a pre-chilled mortar and pestle on an ice bath with sea sand (PanReac AppliChem, Barcelona, Spain) as an abrasive. Then, the homogenate was centrifuged for 5 min at 4500× g at 4 °C, and the collected supernatant was immediately used for analysis.
To measure the extracts 1O2 quenching capacity a competition reaction was carried out using endoperoxide (EP, Invitrotech, Kyoto, Japan) as a singlet oxygen generator and 2,5-diphenyl- 3,4-benzofuran (DPBF, Sigma–Aldrich, Barcelona, Spain) as an UV-Vis absorption probe in a 96-well quartz glass microplate. In each well, 15 µL of the peel extract was mixed with 150 µL of DPBF (0.8 mM solution) and 75 µL of EP (1 mM). The microplate was loaded in dim light and on an ice bath. Absorbance changes of DPBF at 413 nm were monitored during a 90 min reaction at 35 °C using a UV-Vis spectrophotometer microplate reader (SPECTROstar® Omega, BMG Labtech, Offenburg, Germany). α-tocopherol (Sigma–Aldrich, Barcelona, Spain) was used as a standard compound and ethanol:chloroform:water (50:50:1, v:v:v) as a blank. The relative SOAC value for each sample was calculated with the following Formula (2):
Each sample was replicated in 3 wells, and the assay was replicated twice. Results were the mean of the replicates in the 2 microplates (mean ± SE).
To measure the extracts 1O2 quenching capacity a competition reaction was carried out using endoperoxide (EP, Invitrotech, Kyoto, Japan) as a singlet oxygen generator and 2,5-diphenyl- 3,4-benzofuran (DPBF, Sigma–Aldrich, Barcelona, Spain) as an UV-Vis absorption probe in a 96-well quartz glass microplate. In each well, 15 µL of the peel extract was mixed with 150 µL of DPBF (0.8 mM solution) and 75 µL of EP (1 mM). The microplate was loaded in dim light and on an ice bath. Absorbance changes of DPBF at 413 nm were monitored during a 90 min reaction at 35 °C using a UV-Vis spectrophotometer microplate reader (SPECTROstar® Omega, BMG Labtech, Offenburg, Germany). α-tocopherol (Sigma–Aldrich, Barcelona, Spain) was used as a standard compound and ethanol:chloroform:water (50:50:1, v:v:v) as a blank. The relative SOAC value for each sample was calculated with the following Formula (2):
Each sample was replicated in 3 wells, and the assay was replicated twice. Results were the mean of the replicates in the 2 microplates (mean ± SE).
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