Mpw 55

The organic acid and sugar content was determined using the method proposed previously by Wojdyło et al.^{30 (link)} by HPLC–PDA (Waters Co.; Milford, CT, USA) and HPLC-ELSD (PL-ELS 1000; Merck; Hitachi, Japan), respectively. The sample (approx. 3 g of fruits and 1 g of leaves) mixed with distilled water, sonicated (Sonic 6D; Polsonic, Warsaw, Poland) for 15 min and boiled for 30 min, finally sample was centrifuged (MPW-55; Warsaw, Poland) at 12,000xg for 10 min at 4 °C. The supernatant (2.5 mL) was applied onto the Sep-Pak C-18 (1 g, Millipore Waters, Milford, MA, USA) and finally eluted by water to Eppendorf tubes. The extract before analysis was filtered through 0.20 μm hydrophilic PTFE membrane (Millex Simplicity Filter; Merck, Germany). All samples were assayed in triplicate repetition. Results expressed as g per 100 g dry weight (dw).

To obtain an extract rich in polyphenols of P. armeniaca L. leaf powder after freeze-drying was mixed with 50% ethanol, sonicated for 20 min (Sonic 6D; Polsonic, Warsaw, Poland), occasionally mixed, and supernatant was separated by centrifuged for 10 min at 19,000× g (MPW-55; Warsaw, Poland). The residue was reextracted 3–4 times and all obtained fractions were mixed and ethanol was evaporated at 40 °C by a scale rotary evaporator (Hei-VAP Expert, Heidolph; Schwabach, Germany). The obtained aqueous fraction was loaded into a glass column (50 cm; Ø 6 cm) filled with Amberlite polymeric XAD-16 resin previously washed with water. The absorbed aqueous fraction was washed with water (1 mL/min) to remove sugars, organic acids or other ballast substances and then polyphenolic compounds were eluted with ethanol (80 and 100%; 1 mL/min). Collected fractions were mixed and, after evaporation (Hei-VAP Expert, Heidolph; Schwabach, Germany) of alcohol at 40 °C, the obtained fraction was freeze-dried to obtain the powder of polyphenol-rich apricot leaf extract (PrALe).

The analysis of organic acids and carbohydrate was performed as described previously by Wojdyło et al. [14 (link)], using HPLC-PDA (Waters Co.; Milford, CT, USA) and HPLC-ELSD (PL-ELS 1000; Merck; Hitachi, Japan), respectively. The sample (approximately 3 g of fruit) was mixed with distilled water, exposed to ultrasounds (Sonic 6D; Polsonic, Warsaw, Poland) for 15 min, heated at 90–100 °C for 30 min, and finally centrifuged (MPW-55; Warsaw, Poland) at 12,000× g for 10 min at 4 °C. The supernatant (2.5 mL) was injected into a Sep-Pak C-18 cartridge (1 g, Millipore Waters, Milford, MA, USA) and eluted with H₂O into Eppendorf tubes. Before analysis, the extract was filtered through a hydrophilic PTFE membrane (0.20 µm; Millex Simplicity filters; Merck, Germany). The organic acids were analyzed on Polymex IEX H column (8 μm, 250 × 8 mm, Watrex; Prague, Czech Republic) using isocratic elution with 0.9 M sulfuric acid in H₂O for 20 min. The carbohydrates were analyzed on Alltech^® Prevail^TM Carbohydrate ES HPLC Column-W 250 × 4.6 mm, 5 µm (Columbia, MD, USA) using isocratic elution with 70% acetonitrile in H₂O for 20 min. The results were expressed in g per 100 g of d.w.