*2.4. Extraction Procedure*

An amount of 0.2 g of byproduct was weighed and brought into 20 mL of ethanol 96 %. The extracts were obtained by the ultrasound-assisted extraction method involving frequencies ranging from 20 kHz to 2000 kHz for 1h at room temperature. Then, the extracts were centrifuged for 10 min at 10,000 rpm to remove the secondary materials [25].

### *2.5. Determination of Phenol Content*

Total phenol content was determined by the Folin–Ciocalteu method [26]. A total of 50 μL of extract was mixed with 10 μL Folin–Ciocalteu reagent, 90 μL distilled water, and 10 μL of saturated sodium carbonate. The 96-well plates were allowed to stand in the dark for 60 min for color development. Absorbance was measured at 765 nm using a FlexStation 3 UV-Vis (Molecular Devices, GA, USA) Spectrophotometer. A standard curve was prepared by using different concentrations (10–50 μg/mL) of gallic acid in the same condition with samples (R<sup>2</sup> = 0.9966). Total phenolic content was expressed as mg gallic acid equivalent/g of byproduct (mg GAE/g).

### *2.6. Determination of Flavonoid Content*

Total flavonoid content (TFC) was assessed through the AlCl3 method described by Woisky and Salatino [27]. Briefly, 0.1 mL sample/standard solution was mixed with 0.1 mL 10% sodium acetate and 0.12 mL 2.5% AlCI3, the final volume being adjusted to 1 mL with 70% ethanol. The samples were then vortexed and incubated in the dark for 45 min. The absorbances were measured at 430 nm. A standard curve was plotted by using different concentrations (5–200 μg/mL) of quercetin (R<sup>2</sup> = 0.9980). Total flavonoid content was expressed as mg quercetin equivalent/g of byproduct (mg QE/g).

### *2.7. Determination of Antioxidant Activity through DPPH, CUPRAC, FRAP, and ABTS Methods*

DPPH radical scavenging activity was determined based on the reduction in DPPH radical, according to Culetu et al. [28], with slight modifications. The reaction mixture consisted of 1 mL of sample and 6 mL of DPPH radical solution, which was incubated for 20 min in the dark. Then, the absorbance was measured at 517 nm. Antioxidant activity was calculated using a calibration curve (0.0156–0.0625 μg/mL) obtained with Trolox (R<sup>2</sup> = 0.9998). The results were expressed in mg Trolox/g of byproduct.

The CUPRAC method is based on the reduction of a cupric complex, neocuproin, by antioxidants in copper form. Copper ion reduction was performed according to a method described by Celik et al. [29]: 60 μL of sample/standard solutions of different concentrations were mixed with 50 μL CuCl2 (10 mM), 50 μL neocuproin (7.5 mM), and 50 μL ammonium acetate buffer 1 M, pH = 7.00. After 30 min, the absorbance was measured at 450 nm. The stock Trolox solutions required for the calibration curve were 2 mM, and the working concentrations were between 0.125 and 2.0 mM (R<sup>2</sup> = 0.9977). The results were expressed in mg Trolox/g of byproduct.

FRAP assay—the determination of the antioxidant capacity of iron reduction was performed by the method described by Thaipong et al. [30]. The stock solutions included 300 mM acetate buffer (3.1 g C2H3NaO2 3H2O and 16 mL C2H4O2), pH 3.6, 10 mM 2,4,6- tripyridyl-s-triazine (TPTZ) solution in 40 mM HCl, and 20 mM FeCl3 6H2O solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution, and 2.5 mL FeCl3 6H2O solution, and then warming at 37 ◦C before using. After incubation, the absorbance was read at 593 nm. A 1 mM Trolox stock solution was used to plot the calibration curve, the concentration ranging between 25 and 250 μM Trolox (R<sup>2</sup> = 0.9962). The results were expressed in mg Trolox/g of byproduct.

Trolox equivalent antioxidant capacity (TEAC) assay was performed according to Re et al. [31] with slight modifications. A stable stock solution of ABTS+ was produced by mixing a solution of 7 mM ABTS in 2.45 mM potassium persulphate. Then, the mixture was left standing in the dark at room temperature for 12–16 h before use. An ABTS+ working solution was obtained by dilution with ethanol to an absorbance of around of 0.70. The reaction mixture consisted in 20 μL of sample/standard and 180 μL of ABTS+ working solution and was incubated 30 min in the dark. The standard curve was linear between 20 and 200 μM Trolox (R<sup>2</sup> = 0.9975). The results were expressed in mg Trolox/g of byproduct.
