*2.4. Determination of Total Phenolic Compounds, Total Flavonoids, and Vitamins A, C, and E in Pomegranate Peel*

Total phenol content was determined with Folin–Ciocalteu reagent according to Muscolo et al. [25]. Briefly, 500 μL of the aqueous extract was mixed with 250 μL of Folin–Ciocalteu reagent and 2 mL of a 20% Na2CO3 aqueous solution, and the mixture was filled up to 50 mL with deionized water and placed in the dark for 1 h. The absorbance was measured at 765 nm using a UV-Vis Agilent 8453 spectrophotometer (Agilent Technologies, Agilent Technologies, Santa Clara, CA, USA). The results were expressed as mg/L of gallic acid equivalents.

Total flavonoid content was determined according to the colorimetric method as reported in Muscolo et al. [25]. The absorbance was measured at 510 nm using a UV-Vis Agilent 8453 spectrophotometer (Agilent Technologies, Agilent Technologies, Santa Clara, CA, USA). The results were expressed as rutin equivalents (mg/L) using a calibration curve.

Vitamin A was detected as reported in Aremu and Nweze [26]. Absorbance was read at 436 nm and vitamin A was expressed as retinol equivalent (RE).

For vitamin C (ascorbic acid) determination, the method of Davies and Masten [27] was used. Pomegranate powders (0.10 g) were extracted with 10 mL of 3% meta-phosphoric acid—98% acetic acid centrifuged at 2370× *g* (4000 rpm) for 10 min, and the supernatant was used for the determination of ascorbic acid.

For vitamin E (α-tocopherol) analysis, pomegranate powder (0.10 g) was extracted with 10 mL of hexane:isopropanol solution (3:2 *v*/*v*) with agitation for 5 h, and centrifuged at 1330× *g* (3000 rpm) for 10 min. The supernatant was used for the determination of vitamin E [28].

#### *2.5. Protein and Carbohydrate Detection in Pomegranate Peel*

Soluble protein was determined using the Bradford method as reported in Muscolo et al. [25] by using Coomassie Brilliant Blue G-250. The absorbance of each sample was measured at 595 nm using an 1800 UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan). Bovine serum albumin > 99% purity (Sigma) was used as standard, and soluble proteins were estimated as mg BSA/g DW.

The total available carbohydrates were measured using the anthrone method with minor modifications as reported in Muscolo et al. [25]. The amount of available carbohydrates

was calculated using a glucose calibration curve (range of 10–100 mg/mL). The results were reported as mg/g DW.
