The total phenolic content was determined using Folin–Ciocalteu
reagent (FCR), as described by Singelton, Orthofer, and Lamuela-
Raventos (1999).
The experiment was performed in two batches which included three replications for each sample and
standard. Methanolic gallic acid solutions (10–400 mg/l) were used
as standards. In each replicate, 100 ll of the appropriately diluted
sample extract, 100 ll of methanol, 100 ll of FCR and finally 700 ll
of Na2CO3 (20%) were added together and vortexed. The mixture
was incubated for 20 min in the dark and room temperature. After
incubation, the mixture was centrifuged at 13,000 rpm for 3 min.
The absorbance of the supernatant was measured at 735 nm by
spectrophotometer. The total phenolic content was expressed as
gallic acid equivalents (GAE)/100 g dry weight (DW) of the sample