3.9. Antioxidant Activity, Phenol, and Flavonoids

Methanol (70%) was used for extracting essential oil from the specimens. In all treatments, 1 g of powdered specimen was transferred to a falcon pipe. Then, 5 milliliters of solvent were added, and the specimens were preserved on a shaker at 200 rpm for 24 min. They were placed in a centrifuge operating at 6000 rpm for 15 min. In the next stage, the supernatant was collected and stored at −20 °C [53]. The samples were assessed for antioxidant activity using DPPH and spectrophotometric methods. In particular, the reduction in free radicals was a measure of antioxidant activity. To do this, 100 microliters of the prepared extract were added to 5 mL of the control solution (blank) at various densities. The solution was shaken vigorously for 10 s and then stored at room temperature in a dark environment. The absorption values of samples were read at 517 nanometers using a spectrophotometer (Epoch Microplate Spectrophotometer, BioTek Instrument, Winooski, VT, USA). Finally, the following Equation (7) was applied to calculate the antioxidant activity of the samples.

The total phenol content was measured by Folin–Ciocalteu reagent [54]. Accordingly, 100 microliters of each sample extract were mixed with 200 microliters of Folin (50%) and 2000 microliters of distilled water. After 3 min, 1000 microliters of sodium carbonate (20%) was added to the solution, shaken, and stored in a dark room. Then, the absorption of each sample was read using a spectrophotometer at a wavelength of 765 nanometers. In this experiment, the gallic acid standard curve was used for determining the total phenol content in the samples. Total flavonoid content was measured via colorimetry of chloride aluminum. For this purpose, 1 mL of each sample was selected, and 300 microliters of sodium nitrate was added along with 600 microliters of sodium chloride (10%). Six minutes after adding sodium chloride, 4 milliliters of NaOH (1 N) was added. This solution reached 10 mL by adding distilled water, and, finally, the optical absorption was read at 510 nanometers. The quercetin standard curve was used for determining the overall flavonoid content in the sample [55].