2.4. Measurements of total antioxidant activity, total phenolics, and
total carotenoids
The ABTS method was applied to the methanolic extracts as
described by Giao et al. (2007). The ABTS (7 mmol/L) was mixed
with potassium persulfate (2.45 mmol/L) and kept for 12e16 h at
room temperature in the dark. The ABTS solution was diluted with
water to an absorbance of 0.7 (±0.02) at 734 nm. After the addition
of 1.0 mL ABTS solution to 10 mL of sample the mixture the absorbance
reading was made after 6 min. The % inhibition of the sample
was then compared with a standard curve made from the corresponding
readings of ascorbic acid (0.02e0.50 mg/mL). Results
were expressed as mg of ascorbic acid equivalent per g of biomass.
Total phenolic content was determined using the FolinCiocalteu
reagent according the method of Singleton and Rossi
(1965) Briefly 50 mL of methanolic extract, 50 mL Folin-Ciocalteau
reagent, 1 mL of Na2CO3 (75 g/L) and 1.4 mL of ultra-pure water
were added to 1.5 mL microcentrifuge tubes and the samples were
vortexed. The tubes were then left in the dark for 60 min at room
temperature. The absorbance of the sample was read at 750 nm
using gallic acid (0.015e0.50 mg/mL) as a standard. Results were
expressed as mg of gallic acid equivalent per g of biomass.
Total carotenoids quantification was achieved spectrophotometric
at 454 nm, as described by Kimura et al. (1990). The bcarotene
(0.063e4.0 mg/L) was used as standard curve. The total
carotenoids content was expressed based on mg of b-carotene
equivalents per g biomass.