Pomegranate fruits were collected at a natural locality in the East Herzegovina region (south of Bosnia and Herzegovina). The peel was separated from the fruit manually, dried for 4–6 days at room temperature, and grounded in powder. The powdered pomegranate peel (100 g) was extracted with 50% ethanol, using 1:10 as a solid to solvent ratio. Extraction was performed in an ultrasonic bath at 600 °C for 40 min. After filtration, the extract was evaporated (Büchi R-210 rotary evaporator, Flawil, Switzerland), and the raw material was then used for further extraction and characterization. The total phenolic content in the pomegranate peel extract was analyzed spectrophotometrically using the Folin–Ciocalteu method as previously described [42 ]. Gallic acid (0–100 mg/L) was used for the preparation of the calibration curve. The results were expressed as mg of gallic acid equivalents per gram of dry weight. The total tannins content was calculated using the method described in the European Pharmacopoeia (Ph Eur 7.0). The percentage content of tannins is expressed as pyrogallol (%, w/w). The total flavonoid content was measured spectrophotometrically [43 (link)], where catechin was used as a standard for the calibration curve. The total flavonoid content of the extract was expressed as mg catechin equivalents per gram of sample. All results represent the mean of three measurements. The detailed protocol is described in our previous paper [44 (link)]. The pomegranate peel was deposited in Botanical Garden “Jevremovac” University of Belgrade (voucher specimen No. BEOU 17742).
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