To evaluate the concentration of TSPCs and AOA, the samples were
extracted according to the modified methodology of Iqbal, Bhanger,
and Anwar (2005) and Pérez-Jiménez and Saura-Calixto (2005). A
one-gram sample was homogenized with 20 mL of 80% methanol in
a 50 mL Falcon tube and agitated for 1 h at room temperature. After
this period, the sample was centrifuged for 10 min at 3000 rpm, and
the supernatant was separated. To the remaining residue, 20 mL of
80% methanol (pH 2.0) was added, and the same procedure of agitation,
centrifugation and separation of the supernatant was completed.
To this residue, 20 mL of 70% acetone was added, and the same procedure
of agitation, centrifugation and separation of the supernatant
was completed. The three supernatants were mixed and used for
the analysis of TSPCs and AOA. The extractions were performed in
triplicate.