## 2 Materials and Methods
### Methods
#### 2.2.5 Antioxidant Activity Estimation  
* 2,2-diphenyl-1-picryl-hydrazyl (DPPH) method  
The sample extract was prepared according to [PERSON] et al. [21]. First, 3 g of the sample was homogenized with 15 mL of trichloroacetic acid (5%), then 10 mL of chloroform was added. DPPH was dissolved in methanol (0.025 g/L) in order to prepare a fresh solution of radical stock. This solution was measured by spectrophotometer against a blank (at 515 nm) and the absorbance was detected as A0. The prepared sample extract (0.2 mL) was added to the DPPH solution (3.8 mL) and left to react for 10 min. Then, the absorbance was measured and indicated as A10. Measurements were performed in duplicate [22].  
The following formula was used for the calculation of results:  
DPPH in % inhibition = (Absorbance 0 \(-\) Absorbance 10/Absorbance 0) \(\times\) 100  
where Absorbance 0 is at time 0 min (control) and Absorbance 10 is at time 10 min (after sample reaction).  
* 2,20-Azino-Bis (3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS) MethodFirst, 0.1 g of the sample was homogenized in dark vials with 20 mL of ethanol and water (1:1). The sample was extracted (in an ultrasound water bath for 30 min) and then filtrated. Next, 0.007 M ABTS solution (10 mL) was mixed with 0.00245 M potassium persulphate solution (10 mL) and left to react for around 12-16 h. This solution was diluted till its final absorbance (at 735 nm) became 0.7. The prepared sample extract (20 \(\mu\)L) was mixed with the ABTS reaction mixture (1980 \(\mu\)L) and left to react in darkness for 5 min. Then, it was measured by spectrophotometer (at 735 nm) to determine the absorbance [23].  
Calculation of the results were performed according to the following formula:  
\[\text{ABTS [in \%]}=[\text{(ABTS Absorbance -- sample Absorbance)/ABTS Absorbance]}\times 100\]  
where ABTS Absorbance is the absorbance of ABTS solution.  
* Ferric Reducing Antioxidant Power (FRAP) method  
First, 0.1 g of the sample was homogenized in 20 mL of ethanol and water (1:1) in order to prepare for the sample extraction. The extraction was filtered (after 30 min in an ultrasound bath). The filtered extract (180 \(\mu\)L) was incubated with distilled water (300 \(\mu\)L) and 3.6 mL of working solution (acetate buffer + TPTZ + FeCl\({}_{3}\times\) 6H\({}_{2}\)O in a ratio of 10:1:1) in the dark (for 8 min). Then, the absorbance of the solution was measured by spectrophotometer (CE/210, UK) at 593 nm. Torlox was used as the standard; the results were expressed as the amount of Trolox (\(\mu\)mol) in one g of sample [24]. Calibration curve of Trolox (r\({}^{2}>\) 0.99) was used for the calculation of results.  
* Cupric reducing antioxidant capacity (CUPRAC) method  
The CUPRAC method was conducted according to [PERSON] et al. [25]. First, 0.1 g of the sample was sonicated with 20 mL of an ethanol-water mixture (1:1) for 30 min in dark glass vials and then filtered. Next, 1 mL of the extract was mixed with each of the following: 1 mL of 0.01 M Copper (II), 1 mL of 0.0075 M Neocuproin, and 1 mL of NH4 Ac buffer pH = 7.0. After incubation in darkness (for one hour), the absorbance of the samples was measured against a blind sample by spectrophotometer (at 450 nm). Trolox was used for the preparation of a calibration curve and the results were expressed as \(\mu\)mol of Trolox per kilogram of sample.