Folin ciocalteu solution

The pomegranate peel powder was purchased from Packtin, s.r.l. (Reggio Emilia, IT). The plants from which the powder was obtained belong to the Wonderful and Ako cultivars, with an average age of 10 years, grown in organic farming. Fertilizers with very similar values of potassium, phosphorus, and nitrogen (20 g per plant every 2 months) were used. The soil was drained to avoid stagnation. Once produced, the PPP was stored up to 18 months in a cool and dry place.
Nitric acid was purchased from Merck (Darmstadt, Germany). Methanol, acetonitrile, 2-propanol were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Folin–Ciocalteu solution, sodium carbonate, gallic acid, Methanol (HPLC grade), acetonitrile (LC-MS grade), phosphoric acid 85%, Luperox^® TBH70X tert-butyl hydroperoxide solution (TBH) and 20,70-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma–Aldrich (Milan, Italy). The chemicals and solvents were analytical grade reagents or LC-MS grade. CellTiter 96^® Aqueous One Solution Assay (MTS) was purchased from Promega (Milan, Italy). The water used in all experiments was obtained from Millipore Milli-Q (Millipore, Bedford, MA, USA). All sample solutions were stored at −20 °C until analysis and were used within three days of preparation.

The total polyphenol content (TPC) of SBP was determined by testing on a laboratory scale with some modifications in volume and concentration referring to the method of Singh et al. [36 ]. Briefly, 50 μL of the SBP solution (1 mg/mL; DW) was reacted with 100 μL of Na₂CO₃ (2% in DW; Sigma-Aldrich, St. Louis, MO, USA) solution and 50 μL of Folin–Ciocalteu solution (0.2 N in DW; Sigma-Aldrich, St. Louis, MO, USA), mixed, and left in the dark. After 10 min, the absorbance was measured at 720 nm using a spectrophotometer (LABTECH Corp., Ortenberg, Germany), and the results are presented as the gallic acid equivalence value (GAE).

The method of Petchidurai (2019), with minor modifications, was used for the determination of total tannins [25 (link)]. Thus, 0.1 g of extract were dissolved in 10 mL of Folin Ciocalteu solution (Sigma-Aldrich Chemie GmbH, Munich, Germany) diluted with water 1:20 (v/v). The obtained mixture was incubated for 3 min at room temperature, followed by the addition of 2 mL of Na₂CO₃ 35% aqueous solution. The final solution was re-incubated for 30 min before reading the absorbance at 725 nm using a Specord 205 UV-VIS spectrophotometer from Analytik Jena Inc. (Jena, Germany). The total tannin content was expressed on the basis of the tannic acid standard curve, which was indicated in mg/gram in the range 20–200 mg/g.