*3.2. Bioactive Compounds: Total Phenolic and Total Flavonoid Contents*

An "antioxidant extract" was prepared by vortex-mixing 40 mg of the sample with 1 mL of 70 % methanol. The mixture obtained was heated for 30 min at 70 ◦C, and then centrifuged (Eppendorf Centrifuge 5804 R, Hamburg, Germany) at 25,200 rcf for 15 min at 1 ◦C. The supernatant which constituted the "antioxidant extract" was filtered (Spartan filters 0.2 mm) into HPLC amber vials and used for the determination of the total phenolic content, total flavonoid content, and antioxidant activities. A slight modification of the methodology of Singleton and Rossi [26] (1965) was used for quantification of total phenolics as follows: 20 µL of the "antioxidant extract" was mixed with 100 µL of Folin-Ciocalteu phenol reagent (1:10 v/v/in bidistilled H2O) and 80 µL of 7.5% Na2CO3 in a 96-well microplate (Multiskan™ FC Microplate Photometer, Waltham, MA, USA). The microplate was incubated in the dark for 15 min at 45 ◦C. Afterward, the absorbance values against the blank value were recorded at 765 nm in a microplate reader (Multiskan GO Microplate Spectrophotometer, Thermo Scientific, Vantaa, Finland). Simultaneously, gallic acid solutions of different concentrations

were analyzed and a calibration curve was constructed for quantitation purposes. The total phenolic content was expressed as mg gallic acid equivalent (GAE)/g f.w (fresh weight). The total flavonoid content was determined using the colorimetric method described in Dewanto et al. [21] with some modifications. In a 96-well microplate, 25 µL of "antioxidant extract" was combined with 100 µL of water and 10 µL of NaNO2. The microplate was placed in the dark at room temperature. After 5 min, 15 µL of 10% AlCl3 was added to the wells and the microplate was incubated again at room temperature in the dark for 6 min. Then, 50 µL of NaOH 1M and 50 µL of water were added. The absorbance values against the blank value were recorded at 510 nm. Simultaneously, catechin solutions of different concentrations were analyzed. The total flavonoid content was quantified using the calibration curve of catechin and expressed as mg catechin equivalent (CE)/g f.w.
