2.4. Estimation of Polyphenols and Antioxidant Assays

For polyphenol estimation, TPC, TFC and TTC were measured while for antioxidant capacity, three different antioxidant assays, including DPPH, FRAP, and ABTS, were performed using the method of Gu et al. [14]. The data was obtained by the Multiskan^® Go microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA).

The TPC was determined by a spectrophotometric method using Folin–Ciocalteu reagent [15] with some modifications. For determination, 25 µL of the extract was mixed with 25 µL Folin–Ciocalteu reagent solution (1:3 diluted with water) and 200 µL water was added into a 96-well plate (Corning Inc., Midland, NC, USA) followed by incubation at room temperature for 5 min. The reaction mixture was basified by adding 25 µL 10% (w:w) sodium carbonate and incubated again for 60 min in dark area. Then, the absorbance was measured at 765 nm by a spectrophotometer plate reader (Thermo Fisher Scientific, Waltham, MA, USA). The TPC in samples was quantified from a calibration curve prepared with gallic acid standard with different concentrations ranging from 0–200 µg/mL and expressed as mg of gallic acid equivalents (GAE) per g dry weight (dw) (mg GAE/g dw) of the sample.

The TFC was determined by the aluminum chloride method [16] with some modifications. An 80 µL of the extract was mixed with 80 µL of 2% aluminum chloride (diluted with ethanol) and 120 µL of 50 g/L sodium acetate solution in a 96-well plate and incubated at 25 °C for 2.5 h. Then, the absorbance of the mixture was subsequently measured at 440 nm. The TFC was calculated as mg of quercetin equivalent per g (mg QE/g dw) of weight of samples using the calibration curve of quercetin (0–50 µg/mL).

The TTC was determined by vanillin and p-dimethylaminocinnamaldehyde methods [16] with some modifications. Twenty-five µL of the extract was mixed with 150 µL of 4% vanillin solution (diluted with methanol) and 25 µL of 32% sulfuric acid in a 96-well plate and incubated at 25 °C for 15 min and the absorbance was measured at 500 nm. The TTC was expressed as mg of catechin equivalent per g (mg CE/g dw) of samples using a calibration curve prepared with catechin solution ranging from 0–1000 µg/ML.

The DPPH scavenging activity was determined by the DPPH assay method [17] with some modifications. For the DPPH. assay, 40 µL of the extract was added to the 40 µL of DPPH methanolic solution (0.1 mM) in a 96-well plate. The mixtures were shaken vigorously and incubated at 25 °C for 30 min and the absorbance was measured at 517 nm. The DPPH radical-scavenging activity of extracts was expressed as mg of ascorbic acid equivalents per g (mg AAE/g dw) of samples using standard equation, plotted at different concentrations of standard ranges from 0–50 µg/mL.

The FRAP assay was determined based on the method [17] with some modifications. The FRAP method involves assessing the ability of the test material to reduce iron in Fe³⁺-TPTZ complex (ferric-2,4,6-tripyridyl-s-Triazine) to the Fe²⁺-TPTZ complex by the test substance [11]. The FRAP dye was prepared by mixing of sodium acetate solution (300 mM), TPTZ (2, 4, 6-tripyridyl-s-triazine) solution (10 mM), and Fe[III] solution (20 mM) in 10:1:1 ratio, respectively. A 20 µL of extract or standard was added to 280 µL of prepared FRAP dye solution in a 96-well plate and incubated at 37 °C for 10 min. The absorbance was measured at 593 nm. The FRAP results were converted to mg of ascorbic acid equivalents per g (mg AAE/g dw) of samples using the standard curve, plotted at different concentrations of standard ranges from 0–50 µg/mL.

The ABTS scavenging activity was carried out by the ABTS⁺ radical cation decolorization assay with some modification [17]. Here, 5 mL of 7 mmol/L of ABTS solution was mixed with 88 µL of a 140 mM potassium persulfate solution to produce ABTS⁺. The mixture was placed in the dark at room temperature for 16 h. Then, the prepared ABTS⁺ solution was diluted with analytical grade ethanol to obtain an initial absorbance of 0.7 at 734 nm. Then, 10 µL of extract or standard was mixed with 290 µL of prepared diluted ABTS solution in a 96-well plate and incubated at room temperature for 6 min in the dark area. Then, the absorbance was measured at 734 nm after incubation. The antioxidant ability was expressed as mg of ascorbic acid equivalents per g (mg AAE/g dw) of samples using the calibration curve prepared for ascorbic acid, plotted at different concentrations of standard ranges from 0–2000 µg/mL.